Therapeutic and diagnostic tools for impaired glucose tolerance conditions

ABSTRACT

Disclosed herein are novel genes and methods for the screening of therapeutics useful for treating impaired glucose tolerance conditions, as well as diagnostics and therapeutic compositions for identifying or treating such conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.08/857,076, filed Aug. 3, 2000, which is a continued prosecutionapplication of Ser. No. 08/857,076, filed May 15, 1997.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made in part with Government funding, and theGovernment therefore has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] This invention relates to compositions and methods useful fordelaying or ameliorating human diseases associated with glucoseintolerance.

[0004] Diabetes is a major disease affecting over 16 million individualsin the United States alone at an annual cost of over 92 billion dollars.

[0005] Type I diabetes or insulin-dependent diabetes (IDDD) is anautoimmune disease. In the IDDM patient, the immune system attacks anddestroys the insulin-producing beta cells in the pancreas. The centralrole of insulin in human metabolism is to aid in the transport ofglucose into muscle cells and fat cells. The body's inability to produceinsulin results in hyperglycemia, ketoacidosis, thirst, and weight loss.In addition, diabetics often suffer from chronic atherosclerosis andkidney and eyesight failure. A patient with IDDM requires dailyinjections of insulin to survive.

[0006] The most common form of diabetes is non-insulin dependentdiabetes (NIDDM) or Type II diabetes. Type II diabetes is a heterogenousgroup of disorders in which hyperglycemia results from both impairedinsulin secretory response to glucose and decreased insulineffectiveness (i.e., insulin resistance). Older people who areoverweight are at particular risk for Type II diabetes. Genetic studieshave suggested that, Type II diabetes is found in families and that thedisease may be due to multiple genetic defects. In addition, the linkbetween obesity and Type II diabetes is strong. Approximately 80 percentof Type II diabetics are obese. Weight loss and exercise can beeffective to keep blood glucose levels normal, reducing the long-termcomplications of the disease.

[0007] At present there are few reliable methods for presymptomaticdiagnosis of a genetic predisposition for diabetes or obesity. Thesearch for genetic markers linked to diabetes and obesity has provendifficult, and this is especially true for Type II diabetes.

[0008] Treatments for diabetes emphasize control of blood glucosethrough blood glucose monitoring. The majority of patients take oralmedications and/or insulin injections for appropriate control. Treatmentof diabetes is generally chronic and lifelong, and treatments aregenerally not satisfactory over the long run. In addition, insulintreatment may become increasingly ineffective as the disease progresses.While insulin has been known for decades, and within the past decade,the receptors for insulin and aspects of its signaling pathway have beenidentified, the transcriptional output from these signaling pathwayshave not been characterized. In addition, the molecular basis of theobesity-induced insulin resistance is unknown.

SUMMARY OF THE INVENTION

[0009] We have discovered that the C. elegans metabolic regulatory genesdaf-2 and age-1 encode homologues of the mammalian insulin receptor/PI3-kinase signaling pathway proteins, respectively. We have alsodiscovered that the DAF-16 forkhead protein represents the majortranscriptional output of this insulin signaling pathway. For example,we have discovered that it is the dysregulation of the DAF-16transcription factor in the absence of insulin signaling that leads tometabolic defects; inactivation of DAF-16 reverses the metabolic defectscaused by lack of insulin signaling in C. elegans. Finally, we havefound that the C. elegans daf-7, daf-1, daf-4, daf-8, daf-14, and daf-3genes encode neuroendocrine/target tissue TGF-β type signal transductionmolecules that genetically interact with the insulin signaling pathway.Similarly, we have shown that the metabolic defects caused by lack ofneuroendocrine TGF-β signals can be reversed by inactivation of theDAF-3 transcription factor.

[0010] Together, this evidence indicates that the DAF-16, DAF-3, DAF-8,and DAF-14 transcriptional outputs of these converging signalingpathways regulate metabolism. In addition, these discoveries alsoindicate that insulin and TGF-β-like signals are integrated in humans toregulate metabolism, and that the homologues of other DAF proteins arelikely to be defective or down regulated in human diabetic pedigrees aswell as obesity induced diabetes. These results therefore indicate thatthe C. elegans daf genes are excellent candidate genes and proteins forhuman disease associated with glucose intolerance, e.g., diabetes,obesity, and atherosclerosis. Our findings indicate that the humanhomologues of these daf genes and proteins mediate insulin signaling innormal people and may be defective or mis-regulated in diabetics.Moreover, our findings indicate that there are at least two classes oftype II diabetics: those with defects in the TGF-β signaling genes, andthose with defects in insulin signaling genes. Below we describeexemplary sequence and functional characteristics of the humanhomologues of the daf genes.

[0011] The discovery of converging DAF-7 and DAF-2 insulin-likesignaling indicates that many cases of obesity-induced andgenetically-induced diabetes (and obesity) may be treated byadministration of a human DAF-7 polypeptide.

[0012] The discovery that defects in the TGF-β signaling pathway can besuppressed by decreases in DAF-3 pathway activity and that defects inthe insulin pathway can be suppressed by decreases in DAF-16 activityhighlight the utility of transcriptional regulatory DAF proteins in drugdevelopment; in particular, drugs that inhibit the activity of theseproteins are useful for reversing the effects of obesity-induced orgenetically-induced defects in DAF-7 TGF-β type or insulin signaling.

[0013] In one aspect, the invention features a substantially purepreparation of a DAF-2 polypeptide, which can be derived from an animal(for example, a mammal, such as a human, or an invertebrate, such as C.elegans). In preferred embodiments, the DAF-2 polypeptide has insulinreceptor (InR) activity, insulin receptor related activity, insulin-likegrowth factor receptor (IGF-1) receptor activity, or a combination ofthese activities.

[0014] The invention also features isolated DNA encoding a DAF-2polypeptide. This isolated DNA can have a nucleotide sequence thatincludes, for example, the nucleotide sequence of the daf-2 gene shownin FIG. 2B. This isolated DNA can also, in preferred embodiments,complement a daf-2 mutation in C. elegans, an InR mutation in a mouse,or an IGF-1 receptor mutation in a mouse.

[0015] The isolated DNA encoding a DAF-2 polypeptide can be included ina vector, such as a vector that is capable of directing the expressionof the protein encoded by the DNA in a vector-containing cell. Theisolated DNA in the vector can be operatively linked to a promoter, forexample, a promoter selected from the group consisting of daf-2, age-1,daf-16, daf-1, daf-4, daf-3, and akt promoters. The isolated DNAencoding a DAF-2 polypeptide, or a vector including this DNA, can becontained in a cell, such as a bacterial, mammalian, or nematode cell.

[0016] Also included in the invention is a method of producing arecombinant DAF-2 polypeptide, and a DAF-2 polypeptide produced by thismethod. This method involves (a) providing a cell transformed withisolated DNA that (i) encodes a DAF-2 polypeptide, and (ii) ispositioned for expression in the cell, under conditions for expressingthe isolated DNA, and (b) isolating the recombinant DAF-2 polypeptide.

[0017] A substantially pure antibody, such as a monoclonal or polyclonalantibody, that specifically recognizes and binds a DAF-2 polypeptide isalso included in the invention.

[0018] The invention also features a method of detecting a gene, or aportion of a gene, that is found in a human cell and has sequenceidentity to the daf-2 sequence of FIG. 2B. In this method, isolated DNAencoding a DAF-2 polypeptide, a portion of such DNA greater than about12 residues in length, or a degenerate oligonucleotide corresponding toSEQ ID NOS: 33, 34, 79, 80, 81, 82, 83, or 84, is contacted with apreparation of DNA from the human cell under hybridization conditionsthat provide detection of DNA sequences having about 70% or greaternucleic acid sequence identity to the daf-2 sequence of FIG. 2B. Thismethod can also include a step of testing the gene, or portion thereof,for the ability to functionally complement a C. elegans daf-2 mutant.

[0019] Another method included in the invention is a method of isolatinga gene, or a portion of a gene, that is found in a human cell and has atleast 90% nucleic acid sequence identity to a sequence encoding SEQ IDNOS: 33, 34, 79, 80, 81, 82, 83, or 84. This method involves (a)amplifying by PCR the human gene, or portion thereof, usingoligonucleotide primers that (i) are each greater than about 12 residuesin length, and (ii) each have regions of complementarity to opposite DNAstrands in a region of the nucleotide sequence of FIG. 2B, and (b)isolating the human gene, or portion thereof. This method can alsoinclude a step of testing the gene, or portion thereof, for the abilityto functionally complement a C. elegans daf-2 mutant.

[0020] In another aspect, the invention features a substantially purepreparation of a DAF-3 polypeptide, which can be derived from an animal(for example, a mammal, such as a human, or an invertebrate, such as C.elegans). In a preferred embodiment, the polypeptide is a SMAD protein.In other preferred embodiments, the polypeptide is capable of bindingand interacting with a nematode DAF-1, DAF-4, DAF-8, DAF-14, or DAF-16polypeptide.

[0021] The invention also features isolated DNA encoding a DAF-3polypeptide. This isolated DNA can have a sequence that includes, forexample, the nucleotide sequence of a daf-3 gene shown in FIGS. 11A,11B, or 11C. This isolated DNA can also, in preferred embodiments,complement a daf-3 mutation in C. elegans or complement a daf-3 knockoutmouse.

[0022] The isolated DNA encoding a DAF-3 polypeptide can be included ina vector, such as a vector that is capable of directing the expressionof the protein encoded by the DNA in a vector-containing cell. Theisolated DNA in the vector can be operatively linked to a promoter, forexample, a promoter selected from the group consisting of daf-3, daf-4,daf-16, daf-2, age-1, and akt promoters. The isolated DNA encoding aDAF-3 polypeptide, or a vector including this DNA, can be contained in acell, such as a bacterial, mammalian, or nematode cell.

[0023] Also included in the invention is a method of producing arecombinant DAF-3 polypeptide, and a DAF-3 polypeptide produced by thismethod. This method involves (a) providing a cell transformed withisolated DNA that (i) encodes a DAF-3 polypeptide, and (ii) ispositioned for expression in the cell, under conditions for expressingthe isolated DNA, and (b) isolating the recombinant DAF-3 polypeptide.

[0024] A substantially pure antibody, such as a monoclonal or polyclonalantibody, that specifically recognizes and binds a DAF-3 polypeptide isalso included in the invention.

[0025] The invention also features a method of detecting a gene, or aportion of a gene, that is found in a human cell and has sequenceidentity to any of the daf-3 sequences of FIGS. 11A, 11B, or 11C. Inthis method, isolated DNA encoding a DAF-3 polypeptide, a portion ofsuch DNA that is greater than about 12 residues in length, or adegenerate oligonucleotide corresponding to SEQ ID NOS: 35, 36, or 85,is contacted with a preparation of DNA from the human cell underhybridization conditions that provide detection of DNA sequences havingabout 70% or greater nucleic acid sequence identity to any of the daf-3sequences of FIGS. 11A, 11B, or 11C. This method can also include a stepof testing the gene, or portion thereof, for the ability to functionallycomplement a C. elegans daf-3 mutant.

[0026] Another method included in the invention is a method of isolatinga gene, or a portion thereof, that is found in a human cell and has atleast 90% nucleic acid sequence identity to a sequence encoding SEQ IDNOS: 35, 36, or 85. This method includes (a) amplifying by PCR the humangene, or portion thereof, using oligonucleotide primers that (i) areeach greater than about 12 residues in length, and (ii) each haveregions of complementarity to opposite DNA strands in a region of any ofthe nucleotide sequences of FIGS. 11A, 11B, or 11C, and (b) isolatingthe human gene, or portion thereof. This method can also include a stepof testing the gene, or portion thereof, for the ability to functionallycomplement a C. elegans daf-3 mutant.

[0027] In yet another aspect, the invention features a substantiallypure preparation of DAF-16 polypeptide, which can be derived from ananimal (for example, a mammal, such as a human, or an invertebrate, suchas C. elegans). In a preferred embodiment, the polypeptide is a forkheadtranscription factor that binds DNA. In other preferred embodiments, thepolypeptide is capable of interacting with a polypeptide selected fromthe group consisting of DAF-3, DAF-8, and DAF-14.

[0028] The invention also features isolated DNA encoding a DAF-16polypeptide. This isolated DNA can have a sequence that includes, forexample, the sequence of the daf-16 gene shown in FIG. 13A or 13B. Thisisolated DNA can also, in preferred embodiments, complement a daf-16mutation in C. elegans, or complement an FKHR or AFX mutation in amouse.

[0029] The isolated DNA encoding a DAF-16 polypeptide can be included ina vector, such as a vector that is capable of directing the expressionof the protein encoded by the DNA in a vector-containing cell. Theisolated DNA in the vector can be operatively linked to a promoter, forexample, a promoter selected from the group consisting of daf-2, age-1,daf-16, daf-3, daf-4, and akt promoters. The isolated DNA encoding aDAF-16 polypeptide, or a vector containing this DNA, can be contained ina cell, such as a bacterial, mammalian, or nematode cell.

[0030] Also included in the invention is a method for producing arecombinant DAF-16 polypeptide, and a DAF-16 polypeptide produced bythis method. This method involves (a) providing a cell transformed withpurified DNA that (i) encodes a DAF-16 polypeptide, and (ii) ispositioned for expression in the cell, under conditions for expressingthe isolated DNA, and (b) isolating the recombinant DAF-16 polypeptide.

[0031] A substantially pure antibody, such as a monoclonal or polyclonalantibody, that specifically recognizes and binds a DAF-16 polypeptide isalso included in the invention.

[0032] The invention also features a method of detecting a gene, or aportion of a gene, that is found in a human cell and has sequenceidentity to the daf-16 sequence of FIG. 13A or 13B. In this method,isolated DNA encoding a DAF-16 polypeptide, a portion of such DNA thatis greater than about 12 residues in length, or a degenerateoligonucleotide corresponding to SEQ ID NO: 54, 55, 56, or 57, iscontacted with a preparation of DNA from the human cell underhybridization conditions that provide detection of DNA sequences havingabout 70% or greater nucleic acid sequence identity to the daf-16sequence of FIG. 13A or 13B. This method can also include a step oftesting the gene, or portion of the gene, for the ability tofunctionally complement a C. elegans daf-16 mutant.

[0033] Another method included in the invention is a method of isolatinga gene, or a portion of a gene, that is found in a human cell and has atleast 90% nucleic acid sequence identity to a sequence encoding SEQ IDNO: 54, 55, 56, or 57. This method involves (a) amplifying by PCR thehuman gene, or portion thereof, using oligonucleotide primers that (i)are each greater than about 12 residues in length, and (ii) each haveregions of complementarity to opposite DNA strands in a region of thenucleotide sequence of FIG. 13A or 13B, and (b) isolating the humangene, or portion thereof. This method can also include a step of testingthe gene, or portion thereof, for the ability to functionally complementa C. elegans daf-16 mutant.

[0034] In another aspect, the invention features a method of determiningwhether a human gene is involved in an impaired glucose tolerancecondition (for example, a condition involving atherosclerosis) orobesity. This method involves (a) providing a nematode having a mutationin a daf or age gene, and (b) expressing in the nematode the human gene,which is operatively linked to a nematode gene promoter. Complementationof the daf or age mutation in the nematode is indicative of a human genethat is involved in an impaired glucose tolerance condition or obesity.In preferred embodiments, the nematode gene promoter is selected fromthe group consisting of daf-1, daf-3, daf-4, daf-2, age-1, and akt genepromoters. In other preferred embodiments, the daf mutation is selectedfrom the group consisting of daf-2, daf-3, daf-1, daf-4, daf-7, daf-8,daf-11, daf-12, daf-14, and daf-16 mutations. In yet another preferredembodiment, the mutation can also be found in the age-1 gene.

[0035] In further aspects, the invention features methods for diagnosingan impaired glucose tolerance condition (for example, Type II diabetesor a condition involving atherosclerosis), or a propensity for such acondition, in a patient. One such method includes analyzing the DNA ofthe patient to determine whether the DNA contains a mutation in a dafgene. Identification of such a mutation indicates that the patient hasan impaired glucose tolerance condition or a propensity for such acondition. The analysis in this method can be carried out, for example,by nucleotide sequencing or RFLP analysis. The analysis can also includeamplifying (for example, by PCR or reverse transcriptase PCR) the gene(for example, a human gene), or a fragment thereof, using primers, andanalyzing the amplified gene, or a fragment thereof, for the presence ofthe mutation. In preferred embodiments, the daf gene analyzed in thismethod is, for example, a daf-1, daf-2, daf-3 daf-4, daf-7, daf-8,daf-11, daf-12, daf-14, or daf-16 coding sequence, or the daf gene isFKHR or AFX.

[0036] Another method for diagnosing an impaired glucose tolerancecondition, such as Type II diabetes, or a propensity for such acondition, in a patient, includes analyzing the DNA of the patient todetermine whether the DNA contains a mutation in an age gene.Identification of such a mutation indicates that the patient has animpaired glucose tolerance condition or a propensity for such acondition. The analysis in this method can be carried out, for example,by nucleotide sequencing or RFLP analysis. The analysis can also includeamplifying (for example, by PCR or reverse transcriptase PCR) the gene(for example, a human gene), or a fragment thereof, using primers andanalyzing the amplified gene, or fragment thereof, for the presence ofthe mutation. In a preferred embodiment, the age gene is an age-1 codingsequence.

[0037] Yet another method for diagnosing an impaired glucose tolerancecondition, such as Type II diabetes or a condition that involvesatherosclerosis, or a propensity for such a condition, in a patient,includes analyzing the DNA of the patient to determine whether the DNAcontains a mutation in an akt gene. Identification of such a mutationindicates that the patient has an impaired glucose tolerance condition(for example, Type II diabetes) or a propensity for such a condition(for example, a pre-diabetic condition). The analysis in this method canbe carried out, for example, by nucleotide sequencing or RFLP analysis.The analysis can also include amplifying (for example, by PCR or reversetranscriptase PCR) the gene (for example, a human gene), or a fragmentthereof, using primers and analyzing the amplified gene, or fragmentthereof, for the presence of the mutation.

[0038] The invention also includes kits for use in the diagnosis of animpaired glucose tolerance condition, or a propensity for such acondition, in a patient. One such kit includes a PCR primercomplementary to a daf nucleic acid sequence and instructions fordiagnosing an impaired glucose tolerance condition or a propensity forsuch a condition. Another kit includes a PCR primer complementary to anage nucleic acid sequence and instructions for diagnosing an impairedglucose tolerance condition or a propensity for such a condition. Yetanother kit includes a PCR primer complementary to an akt nucleic acidsequence and instructions for diagnosing an impaired glucose tolerancecondition or a propensity for such a condition.

[0039] In another aspect, the invention features methods forameliorating or delaying the onset of an impaired glucose tolerancecondition (for example, Type II diabetes) in a patient. In one suchmethod a therapeutically effective amount of a DAF polypeptide (forexample, the human or nematode DAF-7 polypeptide) is administered to thepatient. In another method, which can be used, for example, in the caseof a condition involving atherosclerosis, a therapeutically effectiveamount of a compound that is capable of inhibiting the activity of aDAF-16 or DAF-3 polypeptide is administered to the patient. In yetanother method, a therapeutically effective amount of a compound thatactivates a DAF-1, DAF-4, DAF-8, DAF-11, or DAF-14 polypeptide isadministered to the patient.

[0040] Another aspect of the invention provides methods for amelioratingor preventing obesity (for example, obesity associated with Type IIdiabetes) in a patient. One such method involves administering to thepatient a therapeutically effective amount of a DAF polypeptide, such asa human or nematode DAF-7 polypeptide. Another such method involvesadministering to the patient a therapeutically effective amount of acompound that is capable of inhibiting the activity of a DAF-16 or DAF-3polypeptide.

[0041] Yet another aspect of the invention features a transgenic,non-human animal, such as a mouse or a nematode, whose germ cells andsomatic cells contain a transgene coding for a mutant DAF polypeptide,for example, a mutant DAF polypeptide that is derived from a human. Inpreferred embodiments, the mutant DAF polypeptide is a DAF-1, DAF-2,DAF-3, DAF-4, DAF-7, DAF-8, DAF-11, DAF-12, DAF-14, or DAF-16polypeptide. In another preferred embodiment, the transgene includes aknockout mutation.

[0042] In a related aspect, the invention features a transgenic,non-human animal, such as a mouse or a nematode, whose germ cells andsomatic cells contain a transgene coding for a mutant AGE polypeptide,for example, a mutant AGE polypeptide derived from a human. In apreferred embodiment, the mutant AGE polypeptide is an AGE-1polypeptide. In another preferred embodiment, the transgene includes aknockout mutation.

[0043] In yet another aspect, the invention features a transgenic,non-human animal, such as a mouse or a nematode, whose germ cells andsomatic cells contain a transgene coding for a mutant AKT polypeptide,for example, a mutant AKT polypeptide derived from a human. In apreferred embodiment, the transgene includes a knockout mutation.

[0044] In related aspects, the invention features cells (for example,cells isolated from a mammal, such as mouse, human, or nematode cells)isolated from the transgenic animals described above.

[0045] The invention also includes methods for producing transgenic,non-human animals. For example, the invention includes a method forproducing a transgenic, non-human animal that lacks an endogenous dafgene and is capable of expressing a human DAF polypeptide. This methodinvolves (a) providing a transgenic, non-human animal whose germ cellsand somatic cells contain a mutation in a daf gene, and (b) introducinga transgene that (i) encodes a human DAF polypeptide, and (ii) iscapable of expressing the human polypeptide, into an embryonal cell ofthe non-human animal.

[0046] Another method included in the invention can be used forproducing a transgenic, non-human animal that lacks an endogenous agegene and is capable of expressing a human AGE polypeptide. This methodinvolves (a) providing a transgenic, non-human animal whose germ cellsand somatic cells contain a mutation in an age gene, and (b) introducinga transgene that (i) encodes a human AGE polypeptide, and (ii) iscapable of expressing the human polypeptide, into an embryonal cell ofthe non-human animal.

[0047] Similarly, the invention includes a method for producing atransgenic, non-human animal that lacks an endogenous akt gene and iscapable of expressing of expressing a human AKT polypeptide. This methodinvolves (a) providing a transgenic, non-human animal whose germ cellsand somatic cells contain a mutation in an akt gene, and (b) introducinga transgene that (i) encodes a human AKT polypeptide, and (ii) iscapable of expressing the human polypeptide, into an embryonal cell ofthe non-human animal.

[0048] Another aspect of the invention features a method of screeningfor a compound that increases the activity of a DAF polypeptide. Thismethod includes (a) exposing a non-human transgenic animal whose germcells and somatic cells contain a transgene coding for a mutant DAFpolypeptide to a candidate compound, and (b) determining the activity ofthe DAF polypeptide in the transgenic animal. An increase in DAFpolypeptide activity, as compared to untreated controls, is indicativeof a compound that is capable of increasing DAF polypeptide activity. Inpreferred embodiments, the compound can be used to treat an impairedglucose tolerance condition or obesity.

[0049] In a related aspect, the invention features a method of screeningfor a compound that decreases the activity of a DAF polypeptide. Thismethod includes (a) exposing a non-human transgenic animal whose germcells and somatic cells contain a transgene coding for a mutant DAFpolypeptide to a candidate compound, and (b) determining the activity ofthe DAF polypeptide in the transgenic animal. A decrease in DAFpolypeptide activity, as compared to untreated controls, is indicativeof a compound that is capable of decreasing DAF polypeptide activity. Inpreferred embodiments, the compound can be used to treat an impairedglucose tolerance condition, obesity, or atherosclerosis. In otherpreferred embodiments, the compound decreases the activity of DAF-3 orDAF-16.

[0050] In another aspect, the invention features a method of screeningfor a compound that increases the activity of an AGE polypeptide. Thismethod includes (a) exposing a non-human transgenic animal whose germcells and somatic cells contain a transgene coding for a mutant AGEpolypeptide to a candidate compound, and (b) determining the activity ofthe AGE polypeptide in the transgenic animal. An increase in AGEpolypeptide activity, as compared to untreated controls, is indicativeof a compound that is capable of increasing AGE polypeptide activity. Inpreferred embodiments, the compound can be used to treat an impairedglucose tolerance condition, obesity, or atherosclerosis.

[0051] In a related aspect, the invention features a method of screeningfor a compound that decreases the activity of a AGE polypeptide. Thismethod includes (a) exposing a non-human, transgenic animal whose germcells and somatic cells contain a transgene coding for a mutant AGEpolypeptide to a candidate compound, and (b) determining the activity ofthe AGE polypeptide in the transgenic animal. A decrease in AGEpolypeptide activity, as compared to untreated controls, is indicativeof a compound that is capable of decreasing AGE polypeptide activity. Inpreferred embodiments, the compound can be used to treat an impairedglucose tolerance condition, obesity, or atherosclerosis. In anotherpreferred embodiment, the AGE polypeptide is AGE-1.

[0052] In another aspect, the invention features a method of screeningfor a compound that increases the activity of an AKT polypeptide. Thismethod includes (a) exposing a transgenic, non-human animal whose germcells and somatic cells contain a transgene coding for a mutant AKTpolypeptide to a candidate compound, and (b) determining the activity ofthe AKT polypeptide in the transgenic animal. An increase in AKTpolypeptide activity, as compared to untreated controls, is indicativeof a compound that is capable of increasing AKT polypeptide activity. Inpreferred embodiments, the compound can be used to treat an impairedglucose tolerance condition, obesity, or atherosclerosis.

[0053] In a related aspect, the invention features a method of screeningfor a compound that decreases the activity of a AKT polypeptide. Thismethod includes (a) exposing a transgenic, non-human animal whose germcells and somatic cells contain a transgene coding for a mutant AKTpolypeptide to a candidate compound, and (b) determining the activity ofthe AKT polypeptide in the transgenic animal. A decrease in AKTpolypeptide activity, as compared to untreated controls, is indicativeof a compound that is capable of decreasing AKT polypeptide activity. Inpreferred embodiments, the compound can be used to treat an impairedglucose tolerance condition or obesity.

[0054] Also included in the invention is a method of screening for acompound that is capable of ameliorating or delaying an impaired glucosetolerance condition. This method involves (a) exposing a transgenic,non-human animal whose germ cells and somatic cells contain a transgenecoding for a mutant DAF, AGE, or AKT polypeptide to a candidatecompound, and (b) monitoring the blood glucose level of the animal. Acompound that promotes maintenance of a physiologically acceptable levelof blood glucose in the animal, as compared to untreated controls, isindicative of a compound that is capable of ameliorating or delaying animpaired glucose tolerance condition. In a preferred embodiment, thecompound can be used to treat Type II diabetes.

[0055] Another method of screening for a compound that is capable ofameliorating or delaying obesity is also included in the invention. Thismethod involves (a) exposing a transgenic, non-human animal whose germcells and somatic cells contain a transgene coding for a mutant DAF,AGE, or AKT polypeptide to a candidate compound, and (b) monitoring theadipose tissue of the animal. A compound that promotes maintenance of aphysiologically acceptable level of adipose tissue in the animal, ascompared to untreated controls, is indicative of a compound that iscapable of ameliorating or delaying obesity.

[0056] A related method of the invention can be used for screening for acompound that is capable of ameliorating or delaying atherosclerosis.This method involves (a) exposing a transgenic, non-human animal whosegerm cells and somatic cells contain a transgene coding for a mutantDAF, AGE, or AKT polypeptide to a candidate compound, and (b) monitoringthe adipose tissue of the animal. A compound that promotes maintenanceof a physiologically acceptable level of adipose tissue in the animal,as compared to untreated controls, is indicative of a compound that iscapable of ameliorating or delaying atherosclerosis.

[0057] In another aspect, the invention includes a method foridentifying a modulatory compound that is capable of decreasing theexpression of a daf gene. This method involves (a) providing a cellexpressing the daf gene, and (b) contacting the cell with a candidatecompound. A decrease in daf expression following contact with thecandidate compound identifies a modulatory compound. In preferredembodiments, the compound can be used to treat an impaired glucosetolerance condition or obesity. In other preferred embodiments, thecompound is capable of decreasing the expression of DAF-3 or DAF-16.This method can be carried out in an animal, such as a nematode.

[0058] In a related aspect, the invention includes a method for theidentification of a modulatory compound that is capable of increasingthe expression of a daf gene. This method involves (a) providing a cellexpressing the daf gene, and (b) contacting the cell with a candidatecompound. An increase in daf expression following contact with thecandidate compound identifies a modulatory compound. In preferredembodiments, the compound can be used to treat an impaired glucosetolerance condition or obesity. In other preferred embodiments, thecompound is capable of increasing expression of DAF-1, DAF-2, DAF-4,DAF-7, DAF-8, DAF-11, or DAF-14. This method can be carried out in ananimal, such as a nematode.

[0059] In another aspect, the invention includes a method for theidentification of a modulatory compound that is capable of increasingthe expression of an age-1 gene. This method involves (a) providing acell expressing the age-1 gene, and (b) contacting the cell with acandidate compound. An increase in age-1 expression following contactwith the candidate compound identifies a modulatory compound. Inpreferred embodiments, the compound is capable of treating an impairedglucose tolerance condition or obesity. This method can be carried outin an animal, such as a nematode.

[0060] In another aspect, the invention provides a method foridentification of a compound that is capable of ameliorating or delayingan impaired glucose tolerance condition. This method involves (a)providing a dauer larvae including a mutation in a daf gene, and (b)contacting the dauer larvae with a compound. Release from the dauerlarval state is an indication that the compound is capable ofameliorating or delaying an impaired glucose tolerance condition. In apreferred embodiment, the dauer larvae carries a daf-2 mutation. Inanother preferred embodiment, the dauer larvae is from C. elegans. Inyet another embodiment, the impaired glucose tolerance conditioninvolves obesity or atherosclerosis.

[0061] In a related aspect, the invention provides a method foridentification of a compound that is capable of ameliorating or delayingan impaired glucose tolerance condition. This method involves (a)providing a dauer larvae including a mutation in an age-1 gene, and (b)contacting the dauer larvae with a compound. Release from the dauerlarval state is an indication that the compound is capable ofameliorating or delaying an impaired glucose tolerance condition. In apreferred embodiment, the dauer larvae carries an age-1 mutation. Inanother preferred embodiment, the dauer larvae is from C. elegans. Inyet another preferred embodiment, the impaired glucose tolerancecondition involves obesity or atherosclerosis.

[0062] In another related aspect, the invention provides a method forthe identification of a compound that is capable of ameliorating ordelaying an impaired glucose tolerance condition. This method involves(a) providing a dauer larvae including a mutation in an akt gene, and(b) contacting the dauer larvae with a compound. Release from the dauerlarval state is an indication that the compound is capable ofameliorating or delaying an impaired glucose tolerance condition. In apreferred embodiment, the dauer larvae is from C. elegans. In anotherpreferred embodiment, the impaired glucose tolerance condition involvesobesity or atherosclerosis.

[0063] In another aspect, the invention provides a method for theidentification of a compound for ameliorating or delaying an impairedglucose tolerance condition. This method involves (a) combining PIP3 andan AKT polypeptide in the presence and absence of a compound underconditions that allow PIP3:AKT complex formation, (b) identifying acompound that is capable of decreasing the formation of the PIP3:AKTcomplex, and (c) determining whether the compound identified in step (b)is capable of increasing AKT activity. An increase in AKT kinaseactivity is taken as an indication of a compound useful for amelioratingor delaying an impaired glucose tolerance condition.

[0064] In yet another aspect, the invention provides a method for theidentification of a compound for ameliorating or delaying an impairedglucose tolerance condition. This method involves (a) providing a daf-7,daf-3 mutant nematode, (b) expressing in the cells of the nematode amammalian DAF-3 polypeptide, whereby the nematode forms a dauer larva,and (c) contacting the dauer larva with a compound. A release from thedauer larval state is an indication that the compound is capable ofameliorating or delaying the glucose intolerance condition.

[0065] In a final aspect, the invention features a method for theidentification of a compound for ameliorating or delaying an impairedglucose tolerance condition. This method involves (a) providing a daf-2,daf-16 mutant nematode, (b) expressing in the cells of the nematode amammalian DAF-16 polypeptide, whereby the nematode forms a dauer larva,and (c) contacting the dauer larva with a compound. A release from thedauer larval state is an indication that the compound is capable ofameliorating or delaying the glucose intolerance condition.

[0066] As used herein, by a “DAF” polypeptide is meant a polypeptidethat functionally complements a C. elegans daf mutation and/or that hasat least 60%, preferably 75%, and more preferably 90% amino acidsequence identity to a 100 amino acid region (and preferably a conserveddomain) of a C. elegans DAF polypeptide. Complementation may be assayedin an organism (for example, in a nematode) or in a cell culture system.Complementation may be partial or complete, but must provide adetectable increase in function (as described herein). DAF polypeptidesare encoded by “DAF” genes or nucleic acid sequences.

[0067] By an “AGE” polypeptide is meant a polypeptide that functionallycomplements a C. elegans age mutation and/or that has at least 60%,preferably 75%, and more preferably 90% amino acid sequence identity toa 100 amino acid region (and preferably a conserved domain) of a C.elegans AGE polypeptide. Complementation may be assayed in an organism(for example, in a nematode) or in a cell culture system.Complementation may be partial or complete, but must provide adetectable increase in a known AGE function. AGE polypeptides areencoded by “AGE” genes or nucleic acid sequences.

[0068] As used herein, by an “AKT” polypeptide is meant a polypeptidethat functionally complements a C. elegans akt mutation and/or thatpossess at least 64% amino acid sequence identity to SEQ ID NO: 60, atleast 71% amino acid sequence identity to SEQ ID NO: 61, at least 79%amino acid sequence identity to SEQ ID NO: 62, at least 63% amino acidsequence identity to SEQ ID NO: 63, at least 48% amino acid sequenceidentity to SEQ ID NO: 64, at least 70% amino acid sequence identity toSEQ ID NO: 65, at least 64% amino acid sequence identity to SEQ ID NO:66, at least 67% amino acid sequence identity to SEQ ID NO: 67, or acombination thereof. Complementation may be assayed in an organism (forexample, in a nematode) or in a cell culture system. Complementation maybe partial or complete, but must provide a detectable increase in aknown AKT function. AKT polypeptides are encoded by “AKT” genes ornucleic acid sequences.

[0069] By a “DAF-2 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-2 mutation and/or that possesses atleast 61% amino acid sequence identity to SEQ ID NO: 33, at least 31%amino acid sequence identity to SEQ ID NO: 34, at least 43% amino acidsequence identity to SEQ ID NO: 79, at least 35% amino acid sequenceidentity to SEQ ID NO: 80, at least 35% amino acid sequence identity toSEQ ID NO: 81, at least 48% amino acid sequence identity to SEQ ID NO:82, at least 43% amino acid sequence identity to SEQ ID NO: 83, at least40% amino acid sequence identity to SEQ ID NO: 84, or a combinationthereof. Preferably, a DAF-2 polypeptide includes an aspartic acid, aproline, a proline, a serine, an alanine, an aspartic acid, a cysteine,or a proline at amino acid positions corresponding to C. elegans DAF-2amino acids 1252, 1312, 1343, 347, 451, 458, 526, 279, and 348respectively, or a combination thereof.

[0070] By a “DAF-3 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-3 mutation and/or that possesses atleast 60% amino acid sequence identity to SEQ ID NO: 35, at least 38%amino acid sequence identity to SEQ ID NO: 36, at least 47% amino acidsequence identity to SEQ ID NO: 85, or a combination thereof.Preferably, a DAF-3 polypeptide includes a proline or a glycine at aminoacid positions corresponding to C. elegans daf-3 amino acids atpositions 200 (proline) and/or 620 (glycine) in FIG. 12A, respectively,or a combination thereof. For example, the polypeptide may include aproline in the motif GRKGFPHV or a glycine in the motif RXXIXXG (where Xis any amino acid).

[0071] By a “DAF-16 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-16 mutation and/or that possesses atleast 71% amino acid sequence identity to SEQ ID NO: 54, at least 35%amino acid sequence identity to SEQ ID NO: 55, at least 65% amino acidsequence identity to SEQ ID NO: 56, at least 53% amino acid sequenceidentity to SEQ ID NO: 57, or a combination thereof. In addition, aDAF-16 polypeptide preferably includes a serine residue in the conservedmotif WKNSIRH (SEQ ID NO: 59).

[0072] By a “DAF-7 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf7 mutation and/or that possesses atleast 29% amino acid sequence identity to SEQ ID NO: 26, at least 66%amino acid sequence identity to SEQ ID NO: 27, at least 45% amino acidsequence identity to SEQ ID NO: 28, at least 33% amino acid sequenceidentity to SEQ ID NO: 29, at least 56% amino acid sequence identity toSEQ ID NO: 30, at least 75% sequence identity to SEQ ID No: 51, or acombination thereof. Preferably, a DAF-7 polypeptide includes a prolineor a glycine at amino acid positions corresponding to C. elegans daf-7amino acids 271 and 280, respectively, or a combination thereof.

[0073] By a “DAF-8 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-8 mutation and/or that possesses atleast 46% amino acid sequence identity to SEQ ID NO: 23, at least 45%amino acid sequence identity to SEQ ID NO: 24, at least 36% amino acidsequence identity to SEQ ID NO: 25, or a combination thereof.

[0074] By an “AGE-1 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans age-1 mutation (previously known as adaf-23 mutation) and/or that possesses at least 40% amino acid sequenceidentity to SEQ ID NO: 17, at least 45% amino acid sequence identity toSEQ ID NO: 18, at least 30% amino acid sequence identity to SEQ ID NO:19, at least 24% amino acid sequence identity to SEQ ID NO: 38, or acombination thereof. Preferably, an AGE-1 polypeptide includes analanine at amino acid positions corresponding to C. elegans age-1 aminoacids 845.

[0075] By a “DAF-1 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-1 mutation and/or that possesses atleast 45% amino acid sequence identity to SEQ ID NO: 13, at least 35%amino acid sequence identity to SEQ ID NO: 14, at least 65% amino acidsequence identity to SEQ ID NO: 15, at least 25% amino acid sequenceidentity to SEQ ID NO: 16, or a combination thereof. Preferably, a DAF-1polypeptide includes a proline at the amino acid position correspondingto C. elegans DAF-1 amino acid 546.

[0076] By a “DAF-4 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-4 mutation and/or that possesses atleast 45% amino acid sequence identity to SEQ ID NO: 20, at least 40%amino acid sequence identity to SEQ ID NO: 21, at least 44% amino acidsequence identity to SEQ ID NO: 22, or a combination thereof.

[0077] By a “DAF-11 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-1 mutation and/or that possesses atleast 40% amino acid sequence identity to SEQ ID NO: 75, at least 43%amino acid sequence identity to SEQ ID NO: 76, at least 36% amino acidsequence identity to SEQ ID NO: 77, at least 65% amino acid sequenceidentity to SEQ ID NO: 78, or a combination thereof.

[0078] By a “DAF-12 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-12 mutation and/or that possesses atleast 42% amino acid sequence identity to SEQ ID NO: 72, at least 58%amino acid sequence identity to SEQ ID NO: 73, at least 34% amino acidsequence identity to SEQ ID NO: 74, or a combination thereof.

[0079] By a “DAF-14 polypeptide” is meant a polypeptide that complements(as defined above) a C. elegans daf-14 mutation and/or that possesses atleast 48% amino acid sequence identity to SEQ ID NO: 68, at least 37%amino acid sequence identity to SEQ ID NO: 69, at least 48% amino acidsequence identity to SEQ ID NO: 70, at least 37% amino acid sequenceidentity to SEQ ID NO: 71, or a combination thereof.

[0080] By “insulin receptor activity” is meant any activity exhibited byan insulin receptor and measured by either (i) activation of insulinreceptor substrate-1 (IRS-1) phosphorylation and recruitment of PI-3kinase, (ii) activation of glucose transporter (Glut 4) fusion with acellular membrane and concomitant glucose uptake, or (iii) activation ofglycogen and/or fat synthesis and concomitant inhibition ofgluconeogenesis or lipolysis or both.

[0081] By “insulin receptor related activity” is meant any activity notdirectly attributable to the insulin receptor but that is measured by anactivation of IRS-1 phosphorylation and recruitment of PI3-kinase.

[0082] By “IGF-1 receptor activity” is meant any activity exhibited byan insulin-like growth factor-1 receptor and measured by (i) activationof IRS-1 phosphorylation and recruitment of PI-3 kinase, (ii) activationof cell division in NIH3T3 cells (e.g., as described in Gronborg et al.,J. Biol. Chem. 268: 23435-23440, 1993), or (iii) activation of bonegrowth in, for example, the mouse model.

[0083] By “SMAD protein” is meant a protein that is capable of couplingto TGF-β type ser/thr receptors. Smad proteins typically contain a smadconserved motif as described by Derynk et al. (Cell 87: 173, 1996).Exemplary smad proteins include, without limitation, DAF-3, MADR-2, MAD,DPC-4, and Sma-2.

[0084] By “AKT activity” is meant any activity exhibited by an AKTpolypeptide and measured by phosphatidylinositol-regulated increases inserine phosphorylation of GSK-3 or activation of non-dauer growth in C.elegans akt mutants.

[0085] By “impaired glucose tolerance condition” is meant any conditionin which blood sugar levels are inappropriately elevated or lack normalmetabolic regulation. Examples of such conditions include, withoutlimitation, Type I diabetes, Type II diabetes, and gestational diabetes,and may be associated with obesity and atherosclerosis.

[0086] By “protein” or “polypeptide” is meant any chain of amino acids,regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation).

[0087] By “substantially pure” is meant a preparation which is at least60% by weight (dry weight) the compound of interest, e.g., any of thepolypeptides of the invention such as the DAF-2, DAF-3, or DAF-16polypeptides or DAF-2, DAF-3, or DAF-16-specific antibodies. Preferablythe preparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight the compound of interest. Purity canbe measured by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

[0088] By “isolated DNA” is meant DNA that is not immediately contiguouswith both of the coding sequences with which it is immediatelycontiguous (one on the 5′ end and one on the 3′ end) in thenaturally-occurring genome of the organism from which it is derived. Theterm therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA or a genomic DNA fragmentproduced by PCR or restriction endonuclease treatment) independent ofother sequences. It also includes a recombinant DNA which is part of ahybrid gene encoding additional polypeptide sequence.

[0089] By a “substantially identical” polypeptide sequence is meant anamino acid sequence which differs only by conservative amino acidsubstitutions, for example, substitution of one amino acid for anotherof the same class (e.g., valine for glycine, arginine for lysine, etc.)or by one or more non-conservative substitutions, deletions, orinsertions located at positions of the amino acid sequence which do notdestroy the function of the polypeptide (assayed, e.g., as describedherein).

[0090] Preferably, such a sequence is at least 75%, more preferably 85%,and most preferably 95% identical at the amino acid level to thesequence used for comparison.

[0091] Homology is typically measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705 or BLAST software available from theNational Library of Medicine). Examples of useful software include theprograms, Pileup and PrettyBox. Such software matches similar sequencesby assigning degrees of homology to various substitutions, deletions,substitutions, and other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

[0092] By a “substantially identical” nucleic acid is meant a nucleicacid sequence which encodes a polypeptide differing only by conservativeamino acid substitutions, for example, substitution of one amino acidfor another of the same class (e.g., valine for glycine, arginine forlysine, etc.) or by one or more non-conservative substitutions,deletions, or insertions located at positions of the amino acid sequencewhich do not destroy the function of the polypeptide (assayed, e.g., asdescribed herein). Preferably, the encoded sequence is at least 75%,more preferably 85%, and most preferably 95% identical at the amino acidlevel to the sequence of comparison. If nucleic acid sequences arecompared a “substantially identical” nucleic acid sequence is one whichis at least 85%, more preferably 90%, and most preferably 95% identicalto the sequence of comparison. The length of nucleic acid sequencecomparison will generally be at least 50 nucleotides, preferably atleast 60 nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides. Again, homology is typically measured usingsequence analysis software (e.g., Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 1710 University Avenue, Madison, Wis. 53705).

[0093] By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of any ofthe polypeptides disclosed herein including, but not limited to, DAF-2,DAF-3, and DAF-16 and any human homolog thereof).

[0094] By “purified antibody” is meant antibody which is at least 60%,by weight, free from the proteins and naturally-occurring organicmolecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, antibody.

[0095] By “specifically binds” is meant an antibody which recognizes andbinds a polypeptide of the invention (e.g., DAF-2, DAF-3, and DAF-16)but which does not substantially recognize and bind other molecules in asample (e.g., a biological sample) which naturally includes apolypeptide of the invention. An antibody which “specifically binds”such a polypeptide is sufficient to detect protein product in such abiological sample using one or more of the standard immunologicaltechniques available to those in the art (for example, Western blottingor immunoprecipitation).

[0096] By “immunological methods” is meant any assay involvingantibody-based detection techniques including, without limitation,Western blotting, immunoprecipitation, and direct and competitive ELISAand RIA techniques.

[0097] By “means for detecting” is meant any one or a series ofcomponents that sufficiently indicate a detection event of interest.Such means involve at least one label that may be assayed or observed,including, without limitation, radioactive, fluorescent, andchemiluminescent labels.

[0098] By “hybridization techniques” is meant any detection assayinvolving specific interactions (based on complementarity) betweennucleic acid strands, including DNA-DNA, RNA-RNA, and DNA-RNAinteractions. Such hybridization techniques may, if desired, include aPCR amplification step.

[0099] By a “modulatory compound”, as used herein, is meant any compoundcapable of either decreasing DAF-3 and DAF-16 expression (i.e., at thelevel of transcription, translation, or post-translation) or decreasingDAF-3 and DAF-16 protein levels or activity. Also included are compoundscapable of either increasing DAF-1, DAF-2, DAF-4, DAF-8, DAF-7, DAF-11,DAF-14, AGE-1, and AKT expression (i.e., at the level of transcription,translation, or post-translation) or increasing DAF-1, DAF-2, DAF-4,DAF-8, DAF-7, DAF-11, DAF-14, AGE-1, and AKT protein levels or theircorresponding activities.

[0100] By “complementation” is meant an improvement of a genetic defector mutation. In one example, complementation of a genetic defect in adaf, age, or akt gene can be carried out by providing the wild-type daf,age, or akt genes, respectively. Complementation is generallyaccomplished by expressing the wild-type version of the protein in ahost cell or animal bearing a mutant or inactive version of the gene.

[0101] Other features and advantages of the invention will be apparentfrom the following detailed description thereof, and from the claims.

DETAILED DESCRIPTION

[0102] The drawings will first be described.

[0103] Drawings

[0104]FIG. 1 shows the genetic and physical map of C. elegans daf-2. Thetop panel shows the genetic map of daf-2. daf-2 maps on the left arm ofchromosome III 11.4 map units to the right of dpy-1 and 1.6 map units tothe left of ben-1 (ACeDB). The middle panel shows the physical map ofdaf-2. daf-2 maps between mgP34 and mgP44 in a region not covered bycosmid clones but covered by YAC Y53G8. Cosmids from the approximatedaf-2 genetic location detect RFLPs between C. elegans strains BristolN2 and Bergerac RC301. mgP31 on cosmid T21A6 is a HindIII RFLP: 5.3 kbin Bristol, 4.5 kb in RC301. mgP33 on cosmid T02B2 is a HindIII RFLP: 9kb in Bristol, 8 kb in RC301. mgP34 on cosmid R10F2 is an EcoRI RFLP:4.1 and 2.8 kb in Bristol, 3.6 kb in RC301. mgP44 on cosmid R07G11 is acomplex EcoRI RFLP: 2.9 kb, 2.4 kb, 1.9 kb and 1.7 kb in Bristol; 3.6kb, 2.5 kb and 1.6 kb in RC301. mgP35 on cosmid T10D5 is a StyI RFLP:5.4 kb in Bristol, 5.8 kb in RC301. mgP32 on cosmid C42B8 is a StyIRFLP: 2.8 kb in Bristol; 2.9 kb in RC301. mgP48 detected with daf-2probe (nt 1277-2126 and 3747-4650) is a HindIII RFLP: 4.3 kb and 7 kb inBristol and 4.1 kb and 6.2 kb in RC301. Thirty-one out of thirty-threeDpy-non-Daf recombinants carry the RC301 allele of mgP34 whereas allthirty-three recombinants in this interval carry the RC301 allele ofmgP44, mapping daf-2 0.69 map units to the right of mgP34 and to theleft of mgP44. Fourteen out of twenty-four Ben-non-Daf recombinantscarry the RC301 mgP44 allele whereas all of these recombinants carry theRC301 allele of mgP34, mapping daf-2 0.66 map units to the left ofmgP44.

[0105] Y53G8 YAC DNA was isolated from CHEF gels as described in Ausubelet al. (Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1990), labeled, and shown to hybridize to multiplerestriction fragments from cosmids bearing mgP34 and mgP44. A probe fromthe insulin receptor homolog on Y53G8 detects the mgP48 RFLP between N2and RC301. All thirty-three Dpy-non-Daf and all twenty-four Ben-non-Dafrecombinants described above carry the RC301 allele of mgP48, indicatingthat daf-2 could not be separated from this insulin receptor gene bythese fifty-seven recombination events in a thirteen map unit interval.

[0106] The bottom panel shows the structure of daf-2 cDNA. The daf-2cDNA was amplified from a cDNA library constructed according to standardmethods by PCR using internal primers derived from the genomic shotgunsequences, vector sequence primers (for 3′ end) and an SL1 transsplicedleader PCR primer (M. Krause, In: Methods Cell Biol., vol. 48, pp.483-512, H. F. Epstein and D. C. Shakes, eds., Academic Press, SanDiego, Calif., 1995). To isolate a cDNA, pooled plasmid DNA from 106clones of a 107 clone complexity cDNA library was used as a PCRtemplate. To obtain a daf-2 cDNA 3′ end, daf-2 internal primerCGCTACGGCAAAAAAGTGAA (SEQ ID NO: 1) in the kinase domain and a cloningvector primer CGATGATGAAGATACCCC (SEQ ID NO: 2) were used in a nestedPCR reaction with adjacent internal primers. For the cDNA fragment fromthe ligand-binding domain to the kinase domain, PCR was carried out withTGATGCGAACGGCGATCGAT (SEQ ID NO: 3) and ACGCTGGATCATCTACATTA (SEQ ID NO:4) primers. For the daf-2 5′ end, SL1 primer GGTTTAATTACCCAAGTTTGAG (SEQID NO: 5) and one internal daf-2 primer GCTCACGGGTCACACAACGA (SEQ ID NO:6) were used in a nested PCR reaction with adjacent internal primers.Using PCR to amplify genomic DNA from a set of 20 daf-2 mutants, wesearched for daf-2 mutations in a 0.8 kb region of the ligand bindingdomain and in a 0.9 kb region of the kinase domain. For sequencing theligand-binding domain PCR primers TGATGCGAACGGCGATCGAT (SEQ ID NO: 7)and TGAGGGCCAACTAAAGAAGAC (SEQ ID NO: 8) were used. In the kinase domainprimers CGCTACGGCAAAAAAGTGAA (SEQ ID NO: 9) and GACGATCCCGAGGTGAGTAT(SEQ ID NO: 10) were used. The presence of an SL1 spliced leadersequence indicates a full length daf-2 cDNA. The predicted ORF is shownas a box; 5′ and 3′ UTRs are shown as thick bars. The predicted DAF-2initiator methionine at base 486 is preceded by an in frame stop codon63 bases upstream. The predicted DAF-2 stop codon is found at base 5658.No consensus polyadenylation signal was found in the cDNA nor in genomicshotgun sequence #00678, which extends 302 bp further downstream. Theinitial insulin receptor homolog shotgun sequences are shown as thinbars above the box.

[0107] Introns were detected by a combination of in silico genomic andcDNA sequence comparison, and by comparison of PCR products derived fromcDNA and genomic DNA templates. The open triangles over a vertical barindicate positions of the detected exon/intron boundaries. All theintron donor sites have GT consensus and the acceptor sites have AGconsensus (Krause, 1995 supra). The triangles without a vertical barindicate the approximate intron locations determined by comparison ofPCR products using genomic DNA or cDNA as a template. Intron lengthswere estimated by comparison of the PCR product size using cDNA orgenomic DNA templates. Genomic regions corresponding to some of theintrons could not be PCR amplified suggesting that these introns arelong. The minimum daf-2 gene size based on this analysis is 33 kb.

[0108]FIG. 2A shows the predicted C. elegans DAF-2 amino acid sequence.The predicted cysteine-rich region (amino acids 207-372) and tyrosinekinase domain (amino acids 1124-1398) are boxed. The signal peptide(amino acids 1-20), proteolysis site (amino acids 806-809),transmembrane domain (amino acids 1062-1085), and PTB binding motif inthe juxtamembrane region (NPEY, amino acids 1103-1106) are underlined.Three DAF-2 tyrosine residues, Y1293, Y1296 and Y1297, in the regioncorresponding to the insulin receptor kinase Y1158 to Y1163 activationloop are likely to be autophosphorylated, based on the predictedsimilarity between the DAF-2 and insulin receptor phosphorylationtargets (FIG. 2B). Another likely target for DAF-2 autophosphorylationis the Y1106 NPEY motif located in the region corresponding to theinsulin receptor juxtamembrane region NPEY motif (at Y972), that hasbeen shown to mediate IRS-1 binding via its PTB domain to the insulinreceptor (White and Kahn, J. Biol. Chem. 269: 1-4, 1994). While DAF-2bears one YXXM motif implicated in coupling to PI 3-kinase, mammalianIRS-1 and Drosophila insulin receptor (Fernandez et al., EMBO J. 14:3373-3384, 1995) bear multiple YXXM motifs. Although no p85-like adaptorsubunit has yet been detected in the C. elegans database, the AGE-1homology to mammalian p110 suggests the existence of a homologous oranalogous adaptor (Morris et al., Nature 382: 536-539, 1996). In theDAF-2 C-terminal domain, two other tyrosine residues may beautophosphorylated and bound to particular SH2-containing proteins:Y1678 binding to a PLC-g or SHP-2 homolog, and Y1686, perhaps binding toSEM-5 (FIG. 2A) (Songyang et al., Cell 72: 767-778, 1993). Whilemutations in, for example, ras and MAP kinase have not been identifiedin screens for dauer constitutive or dauer defective mutations, thesegeneral signaling pathway proteins may couple to DAF-2 as they couple toinsulin signaling in vertebrates (White and Kahn, J. Biol. Chem. 269:1-4, 1994). The predicted phosphotyrosine residues in juxtamembraneregion and the kinase domain activation loop are circled. In theextended C-terminal region, predicted phosphotyrosine residues are alsocircled and SH2-binding sites are underlined (see below).

[0109]FIG. 2B shows the cDNA encoding the C. elegans DAF-2.

[0110]FIG. 2C shows the amino acid comparison of C. elegans DAF-2 to thehuman insulin receptor and human IGF-I receptor (shown in parenthesis),and to the Drosophila insulin receptor homolog, with daf-2 and humaninsulin receptor mutations highlighted. Six daf-2 mutations map in theligand-binding domain: sa187 (C347S, TGT to AGT), e1368 (S451L, TCA toTTA), e1365 (A458T, GCT to ACT), sa229 (D526N, GAT to AAT), and twomutations in mg43 (C279Y, TGT to TAT and P348L, CCC to CTC). Three daf-2mutations substitute conserved amino acid residues in the insulinreceptor kinase domain: sa219 (D1252N, GAT to AAT), e1391 (P1312L, CCCto CTC), and e1370 (P1343S, CCA to TCA). Darkened residues indicateamino acid identity. Hatched residues indicate amino acid similarity.The percentages under the domains represents the percentage of identityobserved between DAF-2 and each receptor. The corresponding BLASTprobabilities of DAF-2 random match to each protein is: 6.4×10⁻¹⁵⁷(human insulin receptor), 2.7×10⁻¹⁵⁶ (human IGF-I receptor), 2.1×10⁻¹⁵³(molluscan InR homolog), 8.3×10⁻¹⁵³ (mosquito InR homolgoue), 1.6×10⁻¹³⁸(human insulin receptor-related receptor), 1.7×10⁻¹²² (Drosophila InRhomolog), 2.0×10⁻¹⁰⁸ (Hydra InR homolog). DAF-2 is more distant from thenext most closely related kinase families: 8.9×10⁻⁵⁸ (v-ros) and3.0×10⁻⁵¹ (trkC neurotrophin receptor).

[0111] Conserved cysteine residues in the ligand-binding domain (top)are marked with dots. In the kinase domain, active site residues thatmediate insulin receptor kinase specificity are marked with stars. Allof these residues are homologous in DAF-2. The mutations found in humanpatients are indicated at the top of the row, and daf-2 allelesubstitutions are indicated below with allele names. The sequencealignments were done with GCG programs, Pileup and Prettybox, and theidentities were calculated with the GCG program, Gap.

[0112]FIG. 3 is a photograph showing the metabolic control by C. elegansdaf-2 and daf-7. The top panel shows low levels of fat accumulation in awild type L3 animal grown at 25° C. that has been stained with Sudanblack. Non-starved animals were fixed in 1% paraformaldehyde in PBS,frozen at −70° C., and freeze-thawed three times. Fixed animals werewashed three times in PBS, and then incubated overnight in 1×Sudan blackaccording to standard methods. The next panel shows higher levels of fataccumulation in daf-2(e1370) grown at the non-permissive temperature of25° C. These animals accumulate fat in both intestinal and hypodermalcells. daf-2(e1370) animals grown at 15° C., the permissive temperature,accumulate low levels of fat, like wild type (data not shown). The nextpanel shows high fat levels in the intestine and hypodermis ofdaf-7(e1372) animals grown at 25° C. The bottom panel shows high levelsof fat in daf-2(e1370) animals grown at the permissive temperature untilthe L4 stage and then shifted to the non-permissive temperature. Thisshows that daf-2 regulates metabolism without entry into the dauerstage.

[0113]FIG. 4 is a schematic diagram showing a model of insulin signalingin the C. elegans dauer formation pathway. In the absence of dauerpheromone, an insulin-like ligand activates DAF-2, and DAF-7 TGF-β-likesignal activates the DAF-1 and DAF-4 receptors. Activated DAF-2autophosphorylates particular tyrosine residues and recruits signalingmolecules, including the PI 3-kinase homolog (a heterodimer of an as yetunidentified p85 homolog and the PI 3-kinase catalytic subunit AGE-1).The AGE-1 PI 3-kinase produces PIP3 second messenger. This secondmessenger may regulate glucose transport (White and Kahn, 1994 supra),metabolic kinase cascades that include AKT and GSK-3 (Hemmings, Science226:1344-1345, 1984; Jonas et al., Nature, 385:343-346, 1997), andtranscription and translation of metabolic genes (White and Kahn, 1994,supra). DAF-16 acts downstream of DAF-2 and AGE-1 in this pathway and isnegatively regulated by them (Vowels and Thomas, Genetics, 130:105-123,1992; Gottlieb and Ruvkun, Genetics, 137:107-110, 1994). While both theDAF-7/TGF-β and DAF-2/insulin signaling pathways converge to controldauer formation, only the DAF-2 pathway controls reproductive phaselongevity. This may be due to non-transcriptional outputs of DAF-2suggested by precedents from insulin receptor signaling. DAF-7 signalingoutput is predicted to be only transcriptional as described herein.

[0114]FIG. 5A shows that C. elegans daf-3 was genetically mapped to aregion on the X chromosome between aex-3 and unc-1. Cosmid and plasmidclones from the region were assayed for transformation rescue (Mello etal., EMBO J 10: 3959-3970,1991). Plasmid pRF4 (rol-6 transformationmarker, 100 ng/ml), and cosmids (5-6 ng/ml) were injected into the gonadof daf-7 (e1372); daf-3 (e1376) animals. Transgenic animals were scoredfor dauer formation at 25° C.; a dauer (i.e., a return to the daf-7phenotype) indicates rescue of daf-3; clones that rescue daf-3 areboxed. B0217 rescues the daf-3 phenotype; eighteen of nineteentransgenic lines were rescued (˜80% dauers). Examination of sequenceprovided by the C. elegans Sequencing Consortium revealed a Smadhomologous gene on B0217. A 13 kb subclone of B0217 containing just theSmad also rescues daf-3 (see FIG. 3). No rescue was seen upon injectionof other cosmids from the region, B0504 (7 lines tested, <1% rescue) andC05H10 (10 lines tested, <1% rescue). mgDf90 is a deletion that removesall of daf-3.

[0115]FIG. 5B shows the structure of the C. elegans daf-3 coding region.The top is the exon/intron structure of daf-3; coding exons are filledboxes, non-coding regions are open boxes, and lines are introns. daf-3cDNAs were isolated according to standard methods. Four cDNAs weresequenced completely; their N-termini are indicated by vertical lines.These three cDNAs contain ˜400 bp of 3′UTR, but no poly-A tail; a C.elegans consensus poly-adenylation sequence is found 12 bp from the 3′end of the cDNAs. The longest of this cDNA appears full-length, as itcontains a methionine codon and the genomic sequence contains no othermethionine codon and no putative splice sites upstream before in-framestop codons. To further characterize the 5′ end of daf-3, PCR productsfrom libraries or individual daf-3 cDNAs were sequenced. From DNAisolated from a cDNA library, we amplified a product with a primer toSL1 and to a region in conserved domain I (shown as primer 1). For theindividual cDNAs, we amplified with a primer to the cDNA vector andprimer 1. These PCR products were sequenced from primer 2 to the 5′ end,and we found that there is alternative splicing at the 5′ end of daf-3,upstream of the conserved domains. The two alternate splice forms areindicated, and the ends of individual cDNAs are indicated by verticallines. Note that the second has the trans-spliced leader SL1 that isfound at the 5′ end of many C. elegans cDNAs; thus, this cDNA shows abonafide 5′ end of daf-3.

[0116]FIG. 5C shows the protein sequence alignment of C. elegans daf-3and the closest homolog found to date, human DPC4, in the Smad conserveddomains I and II. Dots indicate gaps introduced to maximize alignment.DAF-3 is 55% identical to DPC4 in domain I and 30% identical in domainII. daf-3(mg125) and daf-3(mg132) mutations are indicated by boldfaceand underline. The Smad mutational hotspot is underlined. In addition tomg125 and mg132, seven other daf-3 alleles were sequenced in thehotspot; none of them contains a mutation. Alleles sequenced were mg91,mg93, mg105, mg121, mg126, mg133 (isolated by A. Koweek and G.Patterson, unpublished) and sa205.

[0117] FIGS. 6A-6G is a panel of photographs showing C. elegans DAF-3and DAF-4 expression. These photographs show GFP fluorescence, pairedwith DAPI fluorescence or Nomarski optics photographs, as marked. AllDAF-3 photographs show animals with the second plasmid from FIG. 6Aillustrates DAF-3/GFP head expression in an L1 animal. FIG. 6Billustrates DAF-3/GFP expression in the ventral nerve cord of an adultanimal. L1 animals demonstrated similar expression patterns. FIG. 6Cillustrates DAF-3/GFP expression in the intestine of an L1 animal. FIG.6D illustrates DAF-3/GFP expression in the distal tip cell of an L4animal. FIG. 6E illustrates DAF-3/GFP expression in an embryo withapproximately 200 nuclei. FIG. 6F illustrates DAF-4/GFP expression inthe head of an L1 animal. FIG. 6G illustrates DAF-4/GFP expression inthe dorsal nerve cord and ventral nerve cord of an L4 animal.

[0118]FIG. 7 is a table that shows the rescuing ability and suppressionof C. elegans daf-7 by daf-3 plasmids. The solid boxes represent theSmad conserved domains I and II of daf-3; the stippled boxes representgreen fluorescent protein (GFP). For all experiments shown, daf-3plasmids were injected at a concentration of 10 ng/ml, and the pRF4injection marker was injected at a concentration of 90 ng/ml. To scoredauer formation, transgenic adult animals were allowed to lay eggs onplates for several hours at room temperature and were then removed. Theplates were scored after two days at 25° C. The rescue experiment showsthe rescue of daf-7(m62); daf-3(e1376) by each of the fusion proteins.Failure to rescue results in rolling nondauers, while rescue of daf-3results in rolling dauers (the daf-7 phenotype). The control is an arraywith the pRF4 transformation marker and a non-rescuing cosmid. For eachconstruct, four or more lines were measured in two separate experiments.To measure suppression of daf-7, transgenic arrays were crossed intodaf-7 (for plasmids 1 and 3), or produced by injecting directly intodaf-7 (for plasmid 2). Transgenic (rolling) animals were scored forsuppression of daf-7 (=nondauers) or failure to suppress daf-7(=dauers). The controls are two array strains with the pRF4 marker andan unrelated GFP expressing transgene.

[0119]FIG. 8A is a photographs showing that DAF-3/GFP is associated withmetaphase chromosomes. Fixed L1 animals were immunostained with anti-GFPantibody and anti-α-tublin antibody. DNA was visualized using DAPIstaining.

[0120]FIG. 8B is a photograph showing that a truncated C. elegansdaf-3/GFP protein is predominantly nuclear. Wild-type animals wereinjected with the truncated construct shown in FIG. 7 at a concentrationof 10 ng/ml. The pRF4 transformation marker was injected at 100 ng/ml.The photograph shows a late L1 or early L2 animal, and daf-3 ispredominantly nuclear. The clear spot in the center of some of thenuclei is the nucleolus, which has no daf-3/GFP. All cells in theseanimals have predominantly nuclear daf-3/GFP, including the ventral cordneurons, intestinal cells, and distal tip cell (all shown), as well ashead and tail neurons and hypodermal cells.

[0121]FIGS. 9A and 9B show models for the role of the C. elegansdaf-3/DAF-8/DAF-14 Smad proteins in dauer formation. FIG. 9A shows dauerreproductive growth induction. FIG. 9B shows reproductive dauer growthinduction.

[0122]FIG. 10 is a schematic illustration showing the genetic pathwaythat regulates C. elegans dauer formation.

[0123] FIGS. 11A-11C show the cDNA sequences of the differentiallyspliced C. elegans daf-3 transcripts (SEQ ID NOS: 39, 52, and 53).

[0124] FIGS. 12A-12C show the amino acid sequences of the C. elegansDAF-3 polypeptide isoforms (SEQ ID NOS: 40-42).

[0125]FIGS. 13A and 13B show the cDNA sequence of the differentiallyspliced C. elegans daf-16 transcripts (SEQ ID NOS: 43 and 44).

[0126]FIGS. 14A and 14B show the amino acid sequences of the C. elegansDAF-16 polypeptide isoforms (SEQ ID NOS: 45 and 46).

[0127]FIG. 15 shows the cDNA sequence of the C. elegans age-1 gene (SEQID NO: 47).

[0128]FIG. 16 shows the amino acid sequence of the C. elegans AGE-1polypeptide (SEQ ID NO: 48).

[0129]FIG. 17 is a schematic diagram illustrating that convergent TGF-βand insulin signaling activates glucose-based metabolic genes.

[0130]FIG. 18 is a schematic diagram illustrating a switch to fat-basedmetabolism in the absence of DAF-7 and DAF-2 signals (in pheromone).

[0131]FIG. 19 is a schematic diagram illustrating inhibition of theDAF-16 pathway by drugs to ameliorate lack of insulin signaling.

[0132]FIG. 20 is a schematic diagram illustrating inhibition of DAF-3 bydrugs to ameliorate a lack of DAF-7 signaling (for example inobesity-induced diabetes).

[0133]FIG. 21A is an illustration showing that human FKHR and AFX arethe closest relatives to DAF-16. Note that the differentially splicedDAF-16 forkhead domain is less homologous.

[0134]FIG. 21B is an illustration showing a forkhead family tree,illustrating that DAF-16 is much more closely related to FKHR and AFXthan any other forkhead protein.

[0135]FIG. 22 is a photograph showing that daf-16 is expressed in targettissues, like daf-3. This supports the model that DAF-3 and DAF-16 arecapable of interacting.

[0136]FIG. 23 is an illustration showing a model for treatment ofobesity-induced diabetes with DAF-7 protein.

[0137]FIG. 24 is an illustration showing the genetic mapping ofsup(mg144) to the AKT genetic region.

[0138]FIG. 25 is an illustration showing the comparison of C. elegansAKT with mammalian AKT.

[0139]FIG. 26A is a photograph showing the expression of AKT:GFP indaf-2 dauers.

[0140]FIG. 26B is a photograph showing the expression of AKT:GFP in anN2 adult worm.

[0141]FIG. 27 is a schematic illustration showing the molecular map ofdaf-16.

[0142] The DAF-2 Insulin Receptor Family Member Regulates Longevity andDiapause in C. elegans

[0143] Arrest at the C. elegans dauer stage is normally triggered by adauer-inducing pheromone detected by sensory neurons which signal via acomplex pathway to target tissues that are remodeled and metabolicallyshifted such as the germ line, intestine, and ectoderm (Riddle, In:Caenorhabditis elegans II, D. Riddle, T. Blumenthal, B. Meyer, J.Priess, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1997, pp. 739-768. Kenyon, op cit., pp. 791-813.). Genetic epistasisanalysis of daf mutants that arrest at the dauer stage or enter thereproductive life cycle independent of pheromone regulation has revealedparallel genetic pathways that regulate distinct aspects of the dauermetamorphosis (Vowels and Thomas, Genetics 130: 105-123, 1992; Gottlieband Ruvkun, Genetics 137: 107-120, 1994). The pathway that includesdaf-2 is unique in that it controls both reproductive development andnormal senescence: daf-2 mutant animals arrest development at the dauerlarval stage and have dramatically increased longevity (Table I)(Riddle, In: Caenorhabditis elegans II, D. Riddle, T. Blumenthal, B.Meyer, J. Priess, eds., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1997, pp. 739-768; Kenyon, op cit. pp 791-813; Vowels andThomas, Genetics 130: 105-123, 1992; Gottlieb and Ruvkun, Genetics 137:107-120, 1994; Larsen et al., Genetics 139: 1567-1583, 1995; Kenyon etal., Nature 366: 461-464, 1993; Dorman et al., Genetics 141: 1399-1406,1995).

[0144] Table I shows the percentage of dauer formation of daf-2 allelesand the associated mutations. Eggs from animals grown at 15° C. (day 0)were incubated at 15, 20, or 25° C. Numbers in parenthesis are animalscounted. Numbers of wild-type animals and dauers were counted on day 3(20° C. and 25° C.) or day 5 (15° C.). Most of the dauers marked withstars recovered by day 4 (sa229 at 25° C.) or by day 8 (sa229) and sa219at 15° C., e1368 and sg219 at 20° C., and e1365 and e1368 at 25° C.).mg43 was studied as follows: dpy-1(el)daf-2(mg43); SDP3 animals weregrown at 20° C. until the young adult stage. Eggs from five adults werelaid at 15° C. or 20° C. and grown at the same temperatures. Numbers ofDpy-Daf animal and Dpy-non-Daf animals were counted on day 3 (20° C.) orday 5 (15° C.). Sg187 and sg229 were also studied by Malone and Thomas(Genetics 136:879-886, 1994). TABLE I Percentage of dauer formation ofdaf-2 alleles % dauer formation Region Allele mutation 15° C. 20° C. 25°C. cys-rich mg43 C279Y & 100.0 (215) 100.0 (245) n.d. ligand- P348Lbinding sa187 C347S 0.4 (461) 98.7 (224) 100 (910) kinase e1368 S451L0.0 (328) 4.5* (418) 99.7* (698) e1365 A458T 0.0 (450) 0.0 (461) 99.4*(814) sa229 D526N 3.4* (234) n.d. 22.1* (420) sa219 D1252N 10.0* (460)99.7* (396) 100 (514) e1391 P1312L 3.3 (332) 100 (323) 100 (322) e1370P1343S 0.0 (520) 0.0 (188) 100 (635)

[0145] Genetic mapping using both visible genetic markers andrestriction fragment length polymorphism (RFLP) markers places daf-2between mgP34 and mgP44 (FIG. 1). While cosmid coverage of this physicalgenetic region is not complete, YAC Y53G8 carries the genomic regionthat includes mgP34 and mgP44, which flank daf-2 (FIG. 1). As a step inthe C. elegans genome sequencing effort, random M13 subclones derivedfrom Y53G8 were sequenced by the Genome Sequencing Center.

[0146] Sequence Identities Show that DAF-2 is Likely to Bind to anInsulin-like Ligand and to Phoshorylate Tyrosine Residues

[0147] The amino acid sequences and nucleotide sequences encoding DAF-2are shown in FIGS. 2A and 2B, respectively. Using BLASTX to compare 570translated Y53G8 M13 subclone sequences against the Genbank proteindatabase, we found that four sequences are homologous to the mammalianinsulin receptor family. An insulin receptor was a good daf-2 candidategene because insulin regulates vertebrate growth and metabolism (Whiteand Kahn, J. Biol. Chem. 269: 1-4, 1994), and because aphosphatidylinositol (PI) 3-kinase has been shown to act in both theinsulin receptor and daf-2 pathways (White and Kahn, J. Biol. Chem. 269:1-4, 1994; Morris et al., Nature 382: 536-539, 1996). The detection ofmultiple daf-2 mutations in the gene (see below), and the coincidence ofthe genetic location of this insulin receptor homolog with daf-2 (FIG.2C) establish that this insulin receptor homolog corresponds to daf-2.

[0148] The daf-2 transcription unit and gene structure were determinedusing PCR primers derived from daf-2 genomic subclone sequences toamplify daf-2 genomic and cDNA regions. A probable full length daf-2cDNA bears a 5172 base open reading frame, a 485 base 5′UTR and a 159base 3′UTR (FIGS. 1, 2A). The predicted DAF-2 protein shows long regionsof sequence identity to the insulin receptor family. Over the entireprotein, DAF-2 is 35% identical to the human insulin receptor (Ebina etal., Cell 40: 747-58, 1985; Ullrich, et al., Nature 313: 756-61, 1985),34% identical to the human IGF-I receptor (Ullrich, et al., EMBO J.: 5,2503-12, 1986), and 33% identical to the human insulin receptor-relatedreceptor (Shier and Watt, J. Biol. Chem. 264: 14605-8, 1989). DAF-2 isthe only member of the insulin receptor family in the 90 Mb C. elegansgenome sequence (about 90% complete) or in the 10 Mb C. elegans ESTsequence database. Because it is equally distant from insulin, IGF-I,and insulin receptor-related receptors, DAF-2 is probably the homolog ofthe ancestor of these duplicated and diverged receptors, and thus maysubserve any or all of the functions of these mammalian receptors (seebelow). Like these receptors, DAF-2 has a putative signal peptide, acysteine-rich region in the putative ligand binding domain, a putativeproteolysis site, a transmembrane domain, and a tyrosine kinase domain.In addition, DAF-2 has a C-terminal region that may serve a functionsimilar to the mammalian insulin receptor substrate-1 (IRS-1) (FIG. 2;White and Kahn, J. Biol. Chem. 269: 1-4, 1994).

[0149] In the approximately 500 amino acid ligand-binding domain of theinsulin receptor, DAF-2 is 36% identical to insulin receptor and 35%identical to the IGF-I receptor. Twenty-one of twenty-threephylogenetically conserved cysteine residues in this domain areconserved in DAF-2 (FIG. 2C). The DAF-2 cys-rich region is 34% identicalto human insulin receptor and 28% identical to the IGF-I receptor. Sixdaf-2 mutations map in this domain (FIG. 2C, Table I). The mg43 andsa187 mutations substitute conserved residues in the cys-rich region(FIG. 2C). daf-2(mg43) carries two mutations which substitute conservedresidues, which may explain the strength of this allele(non-conditional, Table I). Other substitutions at non-conservedresidues cause less severe phenotypes (Table I). Insulin resistant anddiabetic patients with mutations in the ligand binding domain of thehuman insulin receptor gene have been identified (Taylor, Diabetes 41:1473-1490, 1992) (see below). These mutations impair receptor transportto cell surface, or insulin binding affinity, or both. The DAF-2mutations in this domain might similarly decrease receptor signaling tocause dauer arrest.

[0150] Insulin receptors are α2, β2 tetramers proteolytically processedfrom a single precursor protein (White and Kahn, J. Biol. Chem. 269:1-4, 1994). DAF-2 bears a probable protease recognition site at aposition analogous to the insulin receptor processing site (RVRR806-809) (Yoshimasa et al., J. Biol. Chem. 265: 17230-17237, 1990).

[0151] The 275 amino acid DAF-2 tyrosine kinase domain is 70% similarand 50% identical to the human insulin receptor kinase domain. Uponinsulin binding, the intracellular tyrosine kinase domain of the insulinreceptor phosphorylates particular tyrosine residues flanked bysignature amino acid residues (upstream acidic and downstreamhydrophobic amino acids (Songyang and Cantley, Trends Biochem. Sci. 20:470-475, 1995)) in the intracellular domain as well as on IRS-1 (Whiteand Kahn, J. Biol. Chem. 269: 1-4, 1994). Multiple DAF-2 tyrosineresidues in these sequence contexts are likely autophosphorylationtargets, including three tyrosines in a region similar to the insulinreceptor activation loop and one in the juxtamembrane region asdescribed above (FIG. 2C). Based on the crystal structure of the insulinreceptor kinase domain bound to its activation loop, eight kinase domainresidues mediate target site specificity (Hubbard et al., Nature 372:746-754, 1994). In DAF-2 (but not in more distantly related receptorkinases), these residues are invariant (5/8) or replaced with similaramino acids (3/8: K to R, E to D) (FIG. 2C), suggesting that DAF-2phosphorylates the same target tyrosine motifs as the insulin receptorkinase.

[0152] Three daf-2 missense mutations substitute conserved amino acidresidues in the kinase domain (FIG. 2C, Table I). All three mutationscause moderate to strong dauer constitutive phenotype, but none are asstrong as the non-conditional alleles, for example, mg43 (Table I).Human insulin receptor mutations in the kinase domain exhibit decreasedkinase activity and cause severe insulin resistance and associateddefects (FIG. 2C; Taylor, Diabetes 41: 1473-1490, 1992). Remarkably, ahuman diabetic insulin resistant patient bears the same amino acidsubstitution (P1178L) as daf-2(e1391) (Kim et al., Diabetologia 35:261-266, 1992). This patient was reported to be heterozygous for thissubstitution. daf-2(e1391) is not dominant whereas it is a highlypenetrance recessive mutation (Table I).

[0153] To test for dominance of daf-2(e1391), using a genetically markedbalancer chromosome, 105 dauers segregated from 485 daf-2/+ parents asexpected for a recessive mutations. The genotype of 76/77 of theseanimals was homozygous daf-2(e1391) whereas 1/77 of the dauers wasdaf-2(e1391)/+, indicating a less than 1% dominance. It is possible thatin contrast to C. elegans, the P1178L mutation in humans is dominant, orthat the patient carries a second insulin receptor mutation in trans, orcarries mutations in other genes (for example, other complex type IIdiabetes loci) that enhance the dominance of P1178L (Bruning et al.,Cell 88: 561-572, 1997).

[0154] AGE-1 PI 3-kinase is a Major DAF-2 Signaling Output

[0155] Like the Drosophila insulin receptor homolog, DAF-2 has a longC-terminal extension that may function analogously to mammalian IRS-1(Fernandez et al., EMBO J. 14: 3373-3384, 1995). In mammals, IRS-1tyrosine residues are phosphorylated by the insulin receptor kinase, andthese phosphotyrosines mediate binding to a variety of signalingproteins bearing SH2 domains (White and Kahn, J. Biol. Chem. 269: 1-4,1994; Songyang et al., Cell 72: 767-778, 1993.). Many, but not all, ofthe DAF-2 C-terminal extension tyrosines bear flanking sequence motifssuggestive that they are autophosphorylated (FIG. 2A; Songyang andCantley, Trends Biochem. Sci. 20: 470-475, 1995). Based on precedentsfrom IRS-1 interactions with mammalian PI 3-kinases (White and Kahn, J.Biol. Chem. 269: 1-4, 1994), a YXXM motif at DAF-2 Y1504 is likely tomediate interaction with the AGE-1 PI 3-kinase, which acts in the samegenetic pathway as daf-2 (FIG. 4) (Morris et al., Nature 382: 536-539,1996).

[0156] Three DAF-2 tyrosine residues, Y1293, Y1296 and Y1297, in theregion corresponding to the insulin receptor kinase Y1158 to Y1163activation loop are likely to be autophosphorylated, based on thepredicted similarity between the DAF-2 and insulin receptorphosphorylation targets (FIG. 2C). Another likely target for DAF-2autophosphorylation is the Y1106 NPEY motif located in the regioncorresponding to the insulin receptor juxtamembrane region NPEY motif(at Y972), that has been shown to mediate IRS-1 binding via its PTBdomain to the insulin receptor (White and Kahn, J. Biol. Chem. 269: 1-4,1994). While DAF-2 bears one YXXM motif implicated in coupling to PI3-kinase, mammalian IRS-1 and Drosophila insulin receptor (Fernandez etal., EMBO J. 14: 3373-3384, 1995) bear multiple YXXM motifs. Although nop85-like adaptor subunit has yet been detected in the C. elegansdatabase, the AGE-1 homology to mammalian p110 suggests the existence ofa homologous or analogous adaptor (Morris et al., Nature 382: 536-539,1996). In the DAF-2 C-terminal domain, two other tyrosine residues maybe autophosphorylated and bound to particular SH2-containing proteins:Y1678 binding to a PLC-γ or SHP-2 homolog, and Y1686, perhaps binding toSEM-5 (FIG. 2A) (Songyang et al., Cell 72: 767-778, 1993). Whilemutations in, for example, ras and MAP kinase have not been identifiedin screens for dauer constitutive or dauer defective mutations, thesegeneral signaling pathway proteins may couple to DAF-2 as they couple toinsulin signaling in vertebrates (White and Kahn, J. Biol. Chem. 269:1-4, 1994).

[0157] The insulin receptor also couples to other signaling pathways(White and Kahn, J. Biol. Chem. 269: 1-4, 1994); analogous DAF-2phosphotyrosine residues may mediate these interactions (as describedabove). Thus, we suggest that tyrosines in the DAF-2 cytoplasmic domainare autophosphorylated upon ligand binding, and recruit the AGE-1 PI-3kinase homolog (as well as other molecules) to signal reproductivedevelopment and normal senescence.

[0158] Metabolic Control by daf-2 in Control of Diapause and Aging

[0159] Insulin and its receptor families play key roles in vertebrate(and by our evidence in invertebrates) metabolic and growth control(Kahn and Weir, eds., Joslin's Diabetes Mellitus, Lea & Febiger, 1994).Upon insulin release—by increasing blood glucose and autonomicinputs—insulin receptor engagement directs a shift in the activities ofkey metabolic enzymes, as well as changes in the transcription andtranslation of metabolic regulators in fat, liver, and muscle cells, allof which lead to assimilation of glucose into glycogen and fat (Whiteand Kahn, J. Biol. Chem. 269: 1-4, 1994). IGF-I is released from theliver in response to pituitary growth hormone, and mediates many of thegrowth and development responses to that endocrine signal (Mathews etal., Proc Natl Acad Sci. U.S.A. 83: 9343-7, 1986). Interestingly,lifespan is dramatically increased in dwarf mice with defects in growthhormone signaling, and presumably decreased IGF-I signaling as well(Brown-Borg et al., Nature 384: 33, 1996). No function for the insulinreceptor-related receptor has yet been established, though it isexpressed in conjunction with NGF receptor (Reinhardt et al., J.Neurosci. 14: 4674-4683, 1994).

[0160] Diapause arrest in general and dauer arrest in particular areassociated with major metabolic changes (Tauber et al., SeasonalAdaptation of Insects, Oxford University Press, New York, N.Y., 1986),consistent with a model that daf-2 acts in a metabolic regulatorypathway related to insulin signaling. In wild-type animals, DAF-2signaling allows non-dauer reproductive growth, which is associated withutilization of food for growth in cell number and size, and small storesof fat (FIG. 3). In daf-2 mutant animals, metabolism is shifted to theproduction of fat (FIG. 3) and glycogen (data not shown) in intestinaland hypodermal cells. Even when a temperature-sensitive daf-2 mutantallele is shifted to the non-permissive temperature at the L4 or adultstage (after the critical period for daf-2 control of dauer formation),metabolism is shifted towards storage of fat (FIG. 3). Thus daf-2 alsoregulates metabolism during reproductive development. Similar metabolicshifts are seen in wild-type pheromone-induced dauers (data not shown),age-1 mutants (data not shown), and daf-7 mutants (FIG. 3). In supportof this metabolic shift, in dauer larvae, enzymes that regulateglycolysis are down-regulated while those that regulate glycogen and fatsynthesis are up-regulated, and there is ultrastructural evidence forincreased lipid and glycogen (O'Riordan and Burnell, Comp. Biochem. &Physiol. 92B: 233-238, 1989; O'Riordan and Burnell, Comp. Biochem. &Physiol. 95B: 125-130, 1990; Popham and Webster, Can. J. Zool. 57:794-800, 1978; Wadsworth and Riddle, Develop. Biol. 132: 167-173, 1989).The dauer metabolic shift is associated with arrest of germ lineproliferation, and arrest of somatic cell division and enlargement(Riddle, In: Caenorhabditis elegans II, D. Riddle, T. Blumenthal, B.Meyer, J. Priess, eds., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1997, pp. 739-768; Kenyon, op cit., pp. 791-813).

[0161] There is precedent for insulin-like signaling in invertebratemetabolic and growth control: insulin-like growth factors have beendetected in metabolism-regulating ganglia in molluscs (Roovers et al.,Gene 162: 181-188, 1995) and regulate molting in locust (Hetru et al.,Eur. J. Biochem 201: 495-499, 1991) and silkworm (Kawakami et al.,Science 247: 1333-1335, 1990). Consistent with the daf-2 regulation ofdiapause, injection of insulin into diapausing Pieris brassicae (aninsect) pupae induces recovery (Arpagaus, Roux's Arch. Dev. Biol. 196:527-530, 1987).

[0162] Without being bound to a particular theory, we hypothesize thatan insulin-like signal is up-regulated during reproductive developmentand stimulates DAF-2 receptor autophosphorylation and recruitment of theAGE-1 PI 3-kinase to produce the second messenger PIP3. AGE-1 is likelyto be a major signaling output of DAF-2 because of the similarity of theage-1 and daf-2 mutant phenotypes and because of their similar placementin the epistasis pathway (Vowels and Thomas, Genetics 130: 105-123,1992; Gottlieb and Ruvkun, Genetics 137: 107-120, 1994). Precedents frominsulin receptor signaling suggest the following candidate targets forDAF-2/AGE-1/PIP3 regulation of metabolism: (1) membrane fusion ofvesicles bearing glucose transporters (Kahn and Weir, eds., Joslin'sDiabetes Mellitus, Lea & Febiger, 1994) (or more probably trehalosetransporters (Tauber et al., Seasonal Adaptation of Insects, OxfordUniversity Press, New York, N.Y., 1986)) to facilitate flux of thismolecule for growth and reproductive metabolism; (2) PIP3 activates anAKT/GSK-3 kinase cascade (Hemmings, Science 275: 628-630, 1997) whichmay regulate the activities of glycogen and fat synthetic and lyticenzymes; (3) transcription and translation of metabolic genes such asPEPCK, GDH, fat synthetases, and lipases (White and Kahn, J. Biol. Chem.269:1-4, 1994). Genetic epistasis analysis suggests that DAF-2/AGE-1signaling negatively regulates daf-16 gene activity (Vowels and Thomas,Genetics 130: 105-123, 1992; Gottlieb and Ruvkun, Genetics 137: 107-120,1994). DAF-16 could act at any point downstream of AGE-1 in thissignaling pathway. Evidence is presented herein that DAF-16 representsthe major transcriptional output to DAF-2/AGE-1 PIP3 signaling.

[0163] In addition to these metabolic changes, the DAF-2 signalingcascade also controls the reproductive maturation of the germ line aswell as morphogenetic aspects of the pharynx and hypodermis (Riddle, In:Caenorhabditis elegans II, D. Riddle, T. Blumenthal, B. Meyer, J.Priess, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1997, pp. 739-768; Kenyon, op cit., pp. 791-813). The DAF-2 receptor mayact, for example, in the hypodermal and intestinal target tissues wherewe note a change in metabolism triggered by the dauer regulatory cascade(FIG. 3). It is also possible that DAF-2 regulates the metabolism andremodeling of tissues indirectly, for example, by controlling theproduction of other hormones (Nagasawa et al., Science 226: 1344-1345,1984; Jonas, et al., Nature 385: 343-346, 1997). Expression and geneticmosaic analysis of daf-2 is essential to distinguish these models.

[0164] Even though DAF-2 and the mammalian insulin receptor bothregulate metabolism, the metabolic defects associated with mutations inthese receptors appear to be different. Complete loss of mammalianinsulin receptor activity causes growth arrest at birth (Leprechaunismin humans), and a metabolic shift to runaway lipolysis and ketoacidosis(Kahn and Weir, eds., Joslin's Diabetes Mellitus, Lea & Febiger, 1994),rather than the fat accumulation we observe in daf-2 mutants (FIG. 3).This distinction between insulin receptor and daf-2 mutants may reflectdistinct metabolic responses to this signaling, or a difference betweencomplete loss and declines in insulin signaling. In humans, ketoacidosisis only induced during severe starvation or pathological states wheninsulin levels are very low (Kahn and Weir, eds., Joslin's DiabetesMellitus, Lea & Febiger, 1994). Since none of the daf-2 mutationsdescribed herein are clear null mutations, it is possible that daf-2dauer-constitutive alleles are more analogous to non-null human insulinreceptor mutations. Most daf-2 alleles are temperature sensitive,including alleles isolated in genetic screens that would allow therecovery of non-temperature sensitive mutations (Vowels and Thomas,Genetics 130: 105-123,1992; Gottlieb and Ruvkun, Genetics 137: 107-120,1994). Substitutions of DAF-2 amino acid residues conserved acrossphylogeny cause more penetrant dauer arrest at all temperatures thansubstitutions of non-conserved residues. daf-2 mutants that arrestdevelopment at the dauer stage independent of growth temperature arelikely to have the least gene activity (for example mg43). Several daf-2alleles also cause 5% to 10% embryonic lethality (unpublished results),suggesting that daf-2 functions during embryonic development. None ofthe daf-2 mutations detected so far are nonsense, frameshift, ordeletion alleles. It is possible that the daf-2 null phenotype isstronger than non-conditional dauer arrest, for example embryoniclethality. However, dauer constitutive daf-2 mutant alleles are isolatedfrom EMS mutagenesis at a very high rate (about 1/300 chromosomes),suggesting that the existing alleles are not rare viable alleles. Infact, the 14 year old patient with the same insulin receptor mutation asdaf-2(e1391) was morbidly obese (Kim et al., Diabetologia 35: 261-266,1992), suggesting that metabolic effects of decreased insulin signalingmay be similar to daf-2 mutants.

[0165] It may be significant to human diabetes that animals carryingmutations in daf-16 can grow reproductively even if they also carrydaf-2 and age-1 mutations that disable insulin-like metabolic controlsignals (Vowels and Thomas, Genetics 130:105-123, 1992; Gottlieb andRuvkun, Genetics 137:107-120, 1994). These data suggest that it isunregulated daf-16 gene activity that causes these metabolic shifts. Theanalogous metabolic defects associated with both type I and type IIdiabetes may be caused by similar unregulated activity of the humanDAF-16 homolog. Below we disclose the molecular identity of daf-16.Inhibition of its activity is expected to ameliorate the metabolicdysregulation associated with insulin signaling defects.

[0166] DAF-16 Encodes a Forkhead Transcription Factor Homolog

[0167] Using a combination of genetic mapping and detection of multipledaf-16 mutations in a 5 kb region, we have determined the nucleic acidsequence of daf-16. daf-16 was mapped 1 map unit to the left of lin-11and 3.3 map units right of unc-75 on Chromosome I. This region of thegenome contained a gap that was not covered by cosmids nor YACs. We useda cosmid library (Genome Sciences, Inc.) to walk into the gap. Sequenceanalysis of the ends of four fosmids (H27K20, H01H03, H12I08, andH35K06) revealed that the previously unmapped contig 133 lies in thelin-11 unc-75 gap. Cosmids from the approximate daf-16 genetic locationwere used to detect RFLPs between C. elegans strains Bristol N2 andBergerac RC301: mgP45 on cosmid C39H11, mgP46 on cosmid F28D9, mgP49 oncosmid C35E7, mgP50 is on cosmid C43H8. Zero out of 30 daf non-Uncrecombinants carry the RC301 alleles of mgP45 and mgP50. Two out of 30Daf non-Unc recombinants carry the RC301 allele of mgP49. 10 out of 30Daf non-Unc recombinants carry the RC301 allele of mgP46. 1 out of 4non-Lin Daf recombinants carry the N2 allele of mgP45. 4 out of 4non-Lin Daf recombinants carry the N2 allele of mgP49. These dataindicate that daf-16 lies between cosmids C43H8 and C35E7. The daf-16gene was identified by identifying deletions (mgDf50) and pointmutations (mg53 and mg54) within the forkhead gene on the cosmid R13H8(FIG. 27). There are two major daf-16 transcripts whose sequences areshown in FIG. 13A and FIG. 13B (SEQ ID NOS: 43 and ______,respectively). The amino acid sequences coding for the DAF-16 isoformsare shown in FIGS. 14A-14C (SEQ ID NOS: 44-46).

[0168] We have detected three daf-16 mutations: (1) a large deletion ofconserved regions in daf-16 (mg ΔF50) that proves that the daf-16 nullphenotype is a suppression of daf-2 mutations; (2) an S to Lsubstitution in exon 6 in daf-16 (mg 53) that alters a conserved WKNSIRHmotif; and (3) a nonsense mutation in exon 3 in daf-16 (mg 54) that ispredicted to truncate one of the daf-16 differentially spliced isoforms.Interestingly, this spliced isoform has a distinct forkhead DNA bindingdomain and is therefore expected to bind to distinct promoters orcombinatorial partners. This mutant is a weak suppressor of daf-2,suggesting that both DAF-16 isoforms are necessary for metaboliccontrol.

[0169] Sequence analysis has revealed that DAF-16 is a member of theforkhead (FH) transcription factor family (FIGS. 21A-21B). This strongamino acid homology indicates that DAF-16 is a transcription factor. Ourgenetic analysis indicates that DAF-16 activity is regulated by theDAF-2/AGE-1 insulin signaling pathway. Precedent from another receptorkinase signaling pathway endorses this model:

[0170] the C. elegans LIN-31 forkhead protein has been shown to beregulated by a tyrosine kinase signaling cascade from the LET-23 EGFreceptor homolog (Kim, Genes Dev. 7: 933-947, 1993). Consistent with amodel that DAF-16 acts downstream of insulin signaling, forkheadtranscription factors have also been implicated in metabolic regulation:another FH family member is mammalian HNF-3, an endoderm-specifictranscription factor that acts at the same metabolic control proteinpromoters as HNF-1 and HNF-4, both of which are mutant in maturity onsetdiabetes of the young (MODY) (Yamagata et al., Nature 384: 455-458,1996; Yamagata et al., Nature 384: 458-460, 1996).

[0171] The identification of DAF-16 as a forkhead transcription factoralso explains much of the complex daf genetics of C. elegans. Theconvergence of DAF-7 TGF-β-like signaling and DAF-2 insulin-likesignaling is also explained by our discovery that DAF-16 is a FH proteinand DAF-3 is a Smad protein: Precedent for an interaction between Smadand forkhead proteins has been found in Xenopus. Response to the TGF-βsuperfamily relative activin in early frog development is mediated by aninteraction between the distant relative of DAF-16 called FAST-1, andthe Smad protein, Smad2 (Nature 383: 600-608, 1996). These proteins bindto an enhancer element that is very similar to the myosin II promoter towhich DAF-3 binds (see below). Thus our molecular and genetic dataindicate that the DAF Smad proteins and DAF-16 FH protein interact onmetabolic control promoters.

[0172] Interestingly, analogously to daf-16 bypass of the need for DAF-2insulin receptor signaling in daf-16 mutant animals, lin-31 mutationssuppress the need for LET-23 EGF signaling in C. elegans vulvaldevelopment. These findings indicate that the DAF-2 receptor, adownstream signaling molecule (AGE-1), and a transcription factor targetDAF-16 are involved in insulin-like signaling in C. elegans development.Without being bound by any particular theory, we hypothesize that C.elegans insulin signaling via DAF-2 and AGE-1 activate DAF-16transcriptional activity, so that in a daf-2 or age-1 mutant, or indauer pheromone, DAF-16 acts as a repressor protein causing a metabolicshift to fat metabolism. Our analysis of daf-16 expression shows that,like DAF-3, it is expressed in target tissues (FIG. 22). Our evidenceindicates that Smad protein transcription factors (e.g., DAF 3, DAF8,DAF14) and DAF-16 act on a common set of promoters as combinatorialtranscriptional regulators. Thus, it is at these metabolic genes thatDAF-7 and TGF-β-like and DAF-2 insulin-like signals converge to controlmetabolism. In addition, our evidence indicates that in the presence ofDAF-2 signaling (mimicking high insulin), DAF-16 acts as an activator oftranscription, causing a shift in metabolism toward glucose utilizationfor cell growth. The molecular analysis described herein suggests thatlack of daf-16 gene activity completely bypasses the need for insulinsignaling in metabolic control by releasing metabolic control fromDAF-16 repression. These data suggest that if a human DAF-16 homologacts downstream of insulin signaling in humans, drugs could be developedthat inhibit its activity to bypass the need for insulin signaling.Identification of a such a drug should provide a means for treating bothType I and Type II diabetes.

[0173] As shown in FIGS. 21A-21B, the human FKHR and AFX genes,identified as oncogene breakpoints but not as insulin signaling genes,are much more closely related to DAF-16 than the next closest relativein either Genbank or in the 94% complete C. elegans genome sequence.These data indicate that FKHR and AFX are excellent candidates forsubserving the same function as C. elegans DAF-16: transduction ofinsulin signals and convergence with DAF-7-like Smad signals.

[0174] Evidence for the C. elegans AKT kinase as the probable output ofDAF-2/AGE-1 signaling.

[0175] We screened genetically for mutations that bypass the need forage-1 signaling. This was done by mutagenizing a strain carrying anage-1 (mg44) null mutation (this mutation was heterozygous to allow thestrain to grow). After two generations, animals that could survivewithout age-1 gene activity were selected by their lack of arrest at thedauer stage. We identified daf-16 mutations, as expected. However, wealso identified two new gain of function mutations, sup(mg142) andsup(mg144).

[0176] sup(mg144) suppresses three different age-1 alleles, indicatingthat this mutation bypasses the need for AGE-1 production of PIP3. Forexample, sup(mg144) suppresses the dauer arrest of age-1(mg44), (m333),(mg109) such that fertile adults are formed. sup(mg144) does notsuppress the lack of insulin signaling in the daf-2 mutant:daf-2(e1370); sup(mg144) form dauers at 25 degrees. This suggests thatnot all of the DAF-2 signaling output is via AGE-1. However, in theabsence of both DAF-2 and AGE-1 signaling, sup(mg144) weakly suppresses,allowing some fertile adults to bypass arrest at the dauer stage.daf-2(e1370); sqt-1 age-1(mg44); sup(mg144 )form 8% fertile adults, 12%sterile adults, and 80% dauers at 25 degrees.

[0177] Interestingly, sup(mg144) is a dominant suppressor of age-1mutations. sqt-1 age-1(mg44); sup(mg144)/+ form 100% fertile adults. Thesup(mg144) parental genotype does not affect this outcome. This dataindicates that sup(mg144) is a dominant activating or dominantinactivating mutation.

[0178] Genetic mapping indicates that sup(mg144) may identify anactivating mutation in the C. elegans AKT homologue (FIG. 25). Byplacing sup(mg144) in trans to a multiply marked chromosome (using PCRbased RFLPs), we found that sup(mg144) maps to a 2 map unit geneticinterval that includes C. elegans AKT (FIG. 24).

[0179] 2/39 sup(mg144 ) homozygous animals isolated from asup(mg144)/polymorphic Bergerac chromosome parent recombined betweensup(mg144)mg144 and stP6 (these animals also carried stP18). In thisexperiment mg144 was a het with RW7000 for three generations. So thisplaces sup(mg144) approximately 2.2 mu to the left of stP6).

[0180] 1/39 sup(mg144 ) homozygous animals isolated from asup(mg144)/polymorphic Bergerac chromosome parent recombined betweensup(mg 144) and bP1. In this experiment mg 144 was a het with RW7000 fortwo generations. So this number is approximately 1/80 or 1.2 mu frombP1.

[0181] We generated a GFP fusion to AKT and showed that this gene isexpressed at high levels in dauer larvae but at much lower levels and infewer cells in wild type animals. (FIGS. 26A-26B) Thus AKT represents adauer regulated gene that may respond to DAF-16 and DAF-3transcriptional control. Multiple probable binding sites, related to theDAF-3 binding site in myoII have been identified.

[0182] sup(mg42) identifies another likely output of age-1 signaling

[0183] mg142 suppresses three different age-1 alleles (age-1 (mg44),age-1(m333), and age-1 (mg109) at 20 degrees. age-1(mg44); sup(mg142 )form fertile adults at 15 and 20. At 25 degrees, they form 33% fertileadults and 67% sterile adults.

[0184] sqt-1 age-1(mg44); mg142/+ form 14% fertile adults and 86%sterile adults when the parent was homozygous for mg142. sqt-1age-1(mg44); mg142/+ form 67% fertile adults and 33% sterile adults whenthe parent was heterozygous for mg142. daf-2(e1370); mg142 form sterileadults at 25 degrees; daf-2(e1370); sqt-1 age-1(mg44); mg142 formsterile adults and dauers at 25 degrees. Preliminary mapping placesmg142 approximately 1.6 mu left of unc-1 on LGX

[0185] Diapause and Longevity

[0186] Weak daf-2 and age-1 mutants that do not arrest at the dauerstage nevertheless live much longer than wild-type (Larsen et al.,Genetics 139: 1567-1583, 1995; Kenyon et al., Nature 366: 461-464, 1993;Dorman et al., Genetics 141: 1399-1406, 1995). This connection betweenlongevity and diapause control may not be unique to C. elegans. Diapausearrest is an essential feature of many vertebrate and invertebrate lifecycles, especially in regions with seasonal temperature and humidityextremes (Tauber et al., Seasonal Adaptation of Insects, OxfordUniversity Press, New York, N.Y., 1986). Animals in diapause arrest slowtheir metabolism and their rates of aging, and can survive for periodsfor much longer than their reproductive lifespan (Tauber et al., supra,1986).

[0187] Because insulin-like DAF-2/AGE-1 signaling mediates C. elegansdiapause longevity control, the mammalian insulin signaling pathway mayalso control longevity homologously. In fact, the increase in longevityassociated with decreased DAF-2 signaling is analogous to mammalianlongevity increases associated with caloric restriction (Finch,Longevity, Senescence and the Genome, The University of Chicago Press,Chicago, 1990). It is possible that caloric restriction causes a declinein insulin signaling to induce a partial diapause state, like thatinduced in weak daf-2 and age-1 mutants. The induction of diapause-likestates may affect post-reproductive longevity (Finch, supra), as in C.elegans. Alternatively, it is the changes in the mode and tempo ofmetabolism itself rather than diapause per se that causes increasedlongevity. Another long-lived C. elegans mutant, clk-1, may alsoregulate lifespan via such metabolic effects (Ewbank et al., Science275: 980-983, 1997). This association of metabolic rate with longevityis also consistent with the correlation of free radical generation toaging (Finch, supra).

[0188] Synergistic Control of Metabolism and Diapause by Insulin andTGF-β Signaling Pathways

[0189] In addition to DAF-2 signaling, the DAF-7 TGF-β neuroendocrinesignal is also necessary for reproductive development of C. elegans (Renet al., Science 274: 1389-1391, 1996; Schackwitz et al., Neuron 17:719-728, 1996). The signals in these two pathways are not redundant:animals missing either daf-2 signaling or daf-7 signaling (FIG. 3) shifttheir metabolism and arrest at the dauer stage (Table II). In additionthe phenotypes caused by mutations in either pathway are stronglysynergistic, suggesting that the two pathways are integrated.Synchronised eggs were grown and counted as described above. daf-1(m40)and daf-2(e1370) form 100% dauer at 25° C. Numbers shown in Table IIindicate percentage dauer formation and number of animals counted (inparenthesis). Data presented is the sum of three independent trials.TABLE II Synergy of daf-1 and daf-2 % dauer formation 15° C. 20° C.daf-1 (m40) 0.0 (532) 1.9 (909) daf-2 (e1370) 0.0 (798) 3.8 (503) daf-1(m40); 19.4 (747) 100 (718) daf-2 (e1370)

[0190] This data indicates that DAF-7 TGF-β signals and DAF-2 ligandinsulin-like signals are integrated. In support of this model, weakmutations in the daf-2 insulin signaling pathway and in the daf-7 TGF-βsignaling pathway are highly synergistic (Table II). Genetic epistasisanalysis indicates that the DAF-7 and DAF-2 pathways are parallel ratherthan sequential (Vowels and Thomas, Genetics 130: 105-123, 1992;Gottlieb and Ruvkun, Genetics 137: 107-120, 1994). That is, daf-16mutations strongly suppress daf-2 mutations but not daf-17, daf-1, ordaf-4 mutations, whereas daf-3 mutations strongly suppress daf-17,daf-1, and daf-4 mutations, but not daf-2 mutations. Analogous synergismbetween activin and FGF tyrosine kinase pathways in Xenopus mesoderminduction has been noted (Green et al., Cell 71: 731-739, 1992).

[0191] A dauer-inducing pheromone regulates the production of DAF-7 bythe ASI sensory neuron (Ren et al., Science 274: 1389-1391, 1996;Schackwitz et al., Neuron 17: 719-728, 1996). Because animals carryingdaf-7 nonsense or truncation mutations are responsive to pheromone(Golden and Riddle, Proc. Natl. Acad. Sci. U.S.A. 81: 819-823, 1984), wefurther suggest that the production of the insulin-like ligand forDA-F-2 is also regulated by pheromone. It is not yet clear whether theseDAF-7 and DAF-2 signals converge in target tissues or in otherregulatory (i.e., hormonal) cells; however the expression of the DAF-7receptor pathway genes in essentially all target tissues (infra)suggests that integration occurs there.

[0192] DAF-7 and Diabetes

[0193] Based on the data herein, we propose that in humans as in C.elegans, both a DAF-7-like neuroendocrine signal and insulin arenecessary for metabolic control by insulin. According to this model, thefailure of target tissues to respond to insulin signals in Type IIdiabetic patients could be due to defects either in the insulin orTGF-β-like control pathways. Pedigree analysis has shown a stronggenetic component in Type II diabetes (Kahn et al., Annu. Rev. Med. 47:509-531,1996). In addition, obesity is also a major risk factor in TypeII diabetes (Kahn et al., Annu. Rev. Med. 47: 509-531,1996). Genetic orobesity-induced (Hotamisligil et al., Science 259: 87-91, 1993;Lonnqvist et al., Nat Med 1: 950-953, 1995) declines in a DAF-7-likesignaling pathway could underlie the lack of response to insulin in TypeII diabetes, just as in C. elegans daf-7 mutants cause metabolic defectsvery similar to daf-2 mutants. The discovery that the DAF-7 and DAF-2pathways converge indicates that DAF-7 hormonal signals are defective indiabetic conditions (for example, Type II diabetes), and thatadministration of human DAF-7 is useful for ameliorating the glucoseintolerance, ketoacidosis, and atherosclerosis associated with diabetes.This is shown schematically in FIGS. 17, 18, and 23.

[0194] Whereas the DAF-7 TGF-β like and DAF-2 insulin-like signalingpathways converge to control diapause and metabolism, only theDAF-2/AGE-1 pathway has been implicated in reproductive adult stagelongevity control in the absense of dauer formation (Larsen et al.,Genetics 139: 1567-1583, 1995; Kenyon et al., Nature 366: 461-464, 1993;Dorman et al., Genetics 141: 1399-1406, 1995; and Morris et al., Nature382: 536-539, 1996). Both pathways control the longevity increaseassociated with dauer arrest, since dauer larvae live much longer thanreproductive C. elegans (Riddle, In: Caenorhabditis elegans II, D.Riddle, T. Blumenthal, B. Meyer, J. Priess, ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1997, pp. 739-768; Kenyon, op cit.pp., 791-813: Chayen and Bitensky, Practical Histochemistry, Chichester;New York: Wiley, 1991. The distinction between DAF-7 and DAF-2regulation of longevity could also reflect a more profound regulation ofmetabolism by the DAF-2 pathway than the DAF-7 pathway (FIG. 4). Forexample, based on precedents from TGF-β signaling in other systems andanalysis of this pathway in C. elegans, all of the known signalingoutput of the DAF-7 TGF-β pathway are via downstream Smadtranscriptional regulation (infra). Insulin signaling, and by extension,DAF-2 signaling, is more ramified: outputs from this receptor regulatesugar transport, metabolic enzyme activities, translation of mRNAsencoding these and other enzymes, as well as transcription (White andKahn, J. Biol. Chem. 269: 1-4, 1994). We suggest that it is theregulatory output distinct to the DAF-2 pathway that controls longevity.Alternatively, TGF-β and insulin-like signals may converge only duringthe L1 stage, when diapause is regulated, and that after this stage,only DAF-2 signaling is necessary for normal metabolic control.

[0195] The involvement of insulin and TGF-β signaling in C. elegansdiapause control suggests that the homologous human pathways maysimilarly mediate response to famine. Just as environmental extremes canselect for variation in the genetic pathways that regulate C. elegansdauer formation, famines and droughts in human history may have selectedfor analogous variants in the human homolog of the daf genes. In fact,heterozygous mice carrying either the db or ob recessive diabetes genes,survive fasting about 20% longer than wild type controls (Coleman,Science 203: 663-665, 1979). The high frequency of Type II diabetes inmany human populations may be the legacy of such selections.

[0196] The DAF-3 Smad Protein Anatagonizes DAF-7 TGF-β ReceptorSignaling in the C. elegans Dauer Regulatory Pathway

[0197] In response to environmental signals C. elegans arrestsdevelopment at the anatomically and metabolically distinctivethird-larval dauer stage (Riddle In: C. elegans N, D. L. Riddle, T.Blumenthal, B. J. Meyer, J. R. Priess, eds., Cold Spring Harbor Press,1997, pp. 739-768). Pheromone signal is transduced by chemosensoryneurons (Bargmann and Horvitz, Science 251:1243, 1991) which couple to aTGF-β signaling pathway (Ren et al., Science 274:1389, 1996; Schackwitzet al., Neuron 17:719, 1989), as well as an insulin-related signalingpathway (as discussed, infra) to trigger changes in the development ofthe many tissues remodeled in dauer larvae (Riddle, supra). Mutations indaf-7 (a TGF-β homolog (Estevez et al., Nature 365:644, 1993)), daf-4 (atype II TGF-β receptor (Estevez et al., Nature 365:644, 1993)), daf-1 (atype I TGF-β receptor), daf-8, and daf-14 (Smad homolog) causeconstitutive arrest at the dauer stage even in the absence of pheromone.These genes constitute a neuroendocrine signaling pathway that is activeduring non-dauer development: the DAF-7 TGF-β signal is produced by thesensory neuron ASI during nondauer development, whereas daf-7 expressionin this neuron is inhibited during dauer-inducing conditions (Ren,supra).

[0198] daf-7 and its receptors and Smad proteins are antagonists todaf-3. The dauer constitute phenotypes of mutations in the daf-7 signaltransduction pathway genes (including putative null mutations) are fullysuppressed by mutations in daf-3. These genetic data indicate that inthe absence of daf-7 signaling, daf-3 acts to induce dauer arrest.

[0199] To discern the molecular basis of the DAF-3 function in thispathway, we determined the sequence and expression pattern of daf-3.Cosmids in the daf-3 genetic region were assayed for gene activity bytransformation. Cosmid B0217 partially complemented a daf-3 mutation,while other cosmids from the region did not (FIG. 5A). A subclone ofB0217 containing only the Smad homolog, but no other coding regions alsorescued daf-3. Our detection of mutations in the Smad homolog (seebelow) confirmed its assignment to daf-3. Analysis of daf-3 cDNAsrevealed that the gene was transcribed from fifteen exons and wasalternatively spliced upstream of the region conserved in Smad proteins.(FIG. 5B) The biological activity of these alternatively splicedisoforms is unknown. The nucleotide (SEQ ID NO: 11) and amino acidsequences (SEQ ID NO: 12) of DAF-3 are shown in FIGS. 11 and 12,respectively.

[0200] Thus far, the C. elegans DAF-3 Smad protein is most closelyrelated in sequence to DPC4, which is a putative cofactor for Smad1,Smad2, and Smad3 (Zhang et al., Nature, 383:168, 1996; Lagna et al.,Nature, 383:832, 1996; Savage et al., Proc. Natl. Acad. Sci., 93:790,1996; Hahn et al., Science, 271:350 (1996). Smads have two conserveddomains (Wrana et al., Trends Genet., 12:493, 1996). DAF-3 has these twodomains; compared to its closest known relative DPC-4, daf-3 has 55%amino acid identity in domain I and 30% in domain II (FIG. 5C). However,DPC-4 is not the mammalian DAF-3 homologue: C. elegans Sma-4, forexample, is more closely related to DPC-4 than DAF-3.

[0201] We identified three mutations in daf-3, all of which wereisolated as suppressors of daf-7(e1372). mgDf90 is a homozygous viabledeletion of 15-90 kb that removes the entire Smad gene (FIG. 5A). mgDf90was identified as a spontaneous mutation that suppressed daf-7 in thestrain of GR1300 (daf-7 (e1372) 111; mut-6(st 702) unc-22 (St192) IV).Thus, suppression of the daf-7 dauer constitutive phenotype of daf-3 isdaf-3 null phenotype, demonstrating that wild-type DAF-3 actsantagonistically to signaling from the DAF-7 TGF-β pathway signaling.daf-3(mg125) and daf-3(mg132) are missense mutations that alterconserved residues in domains 1 and 2 respectively (FIG. 5C). Most ofthe mutations detected in other Smads localize to a 45 amino acidsegment of domain II (Wrana et al., Trends in Genet. 12:493, 1996).Clustering of mutations is observed even in DPC4, for which homozygousnull mutations have been identified (Hahn et al., Science 271:350,1996), so the clustering is unlikely to be due to selection for non-nullmutations. This hotspot region was sequenced in nine daf-3 alleles, andno mutations were detected. This difference in mutation location may bea simple statistical anomaly, or may indicate functional differencesbetween DAF-3 and other Smad proteins, consistent with the fact thatDAF-3 is antagonized, rather than activated, by an upstream TGF-βmolecule.

[0202] To determine where DAF-3 may function in control of dauerformation, we examined the expression pattern of a functionaldaf-3/Green Fluorescent Protein (GFP) fusion gene. This was accomplishedby replacing a AvrII/SacI fragment from pGP8 with a PCR product in whichseveral restriction sites were inserted after the last codon of daf-3before the stop codon. A GFP/unc-54 3′ end PCR product from pPD95.81 wascloned into the 3′ restriction sites to produce pGP19. This DAF-3/GFPfusion partially rescues a daf-3 mutant (FIG. 7). GFP fluorescencetherefore indicates the functional location of DAF-3. DAF-7 signalingfrom the ASI neuron begins during the L1 stage, and neuron ablations anddauer-formation assays in various environmental conditions indicate thatthe signal for dauer formation is also received during the first twolarval stages (Ren et al., Science 274:1389, 1996, Schackwitz et al.,Neuron 17:719, 1996; Bargmann and Horvitz, Science 251:1243, 1991;Golden and Riddle, Developmental Biology 102:368, 1984; Swanson andRiddle, Developmental Biology 84:27, 1981). Therefore, we mostextensively examined L1 larvae.

[0203] Almost every transgenic animal showed strong daf-3/GFP expressionin head neurons (FIG. 6A), the ventral nerve cord (both cell bodies andprocesses, see FIG. 6B), the intestinal cells (FIG. 6C), especially themembrane adjacent to the intestinal lumen, the tail hypodermis, and tailneurons. For all GFP scoring, animals were grown at 25-26° C. Forscoring of DAF-3/GFP in wild-type and in dauer constitutive mutantbackgrounds, three or more lines were scored in each case. A largenumber of animals were surveyed to determine the expression pattern, andat least 30 animals were scored head-to-tail, and expression was talliedfor each tissue. About half of the transgenic animals have weakexpression in V blast cells, P blast cells, hyp7 hypodermal cells, andthe pharynx. The weak expression impedes cell identification, but themain body of the pharynx is filled, implying expression in pharyngealmuscle (FIG. 6A). Expression is rarely detected in dorsal body wallmuscle. The expression pattern in older larvae and adults is similar tothat of L1 animals. In addition, DAF-3/GFP is expressed in the distaltip cells and in their precursors, Z1.a and Z4.p, throughout development(FIG. 6D, FIG. 8). DAF-3/GFP is also strongly expressed in unidentifiedvulval cells. In wild-type embryos of 200-400 cells, DAF-3/GFP isexpressed uniformly thoughout the embryo (FIG. 6E). Under the conditionsof the experiment, which promote reproductive growth, the subcellularlocalization of the DAF-3/GFP protein is mainly cytoplasmic (FIGS. 6B-E,and see below).

[0204] Because DAF-3 activity may be regulated by the DAF-1 and DAF-4TGF-β receptors, we examined the expression of a DAF-4/GFP fusion inwild-type (FIGS. 6A-6G). This construct complements a daf-4 mutant. A 10kb SalI fragment from cosmid CO5D2 contains 3 kb of sequence upstream ofthe daf-4 transcriptional start, and all of the daf-4 coding regionexcept codons for the last fourteen residues of daf-4. This fragment wassubcloned into the SalI site of the GFP plasmid TU#61 (Chalfie et al.,Science 263: 802-805, 1994). This plasmid was injected into thedaf-4(m72) strain to test the fusion for DAF-4 activity. More than 95%of the transgenic animals were rescued for the dauer-constitutive andsmall phenotypes of daf-4(m 72), indicating that the fusion has robustDAF-4 activity. The pattern of DAF-4/GFP expression is similar to thatof daf-3/GFP, except that DAF-4/GFP is localized to membranes,consistent with its role as a receptor. DAF-4/GFP is expressed morestrongly in the pharynx (FIGS. 6F-G), and more weakly in the ventralnerve cord cell bodies and the body hypodermis. Expression of DAF-4/GFPin wild-type animals is detected later than DAF-3/GFP. DAF-4/GFP isfirst detectable at late embryogenesis when the embryo resembles an L1larva. The DAF-4/GFP construct contains an older version of GFP than inDAF-3/GFP; in the older version, the chromophore takes longer to mature.To verify that the difference in embryonic expression of DAF-4/GFP andDAF-3/GFP is not an artefact of the slower maturation time in the daf-4strain, we used anti-GFP antibodies to assay GFP. These antibodiesshould recognize the two forms of GFP equally well. We found that theantibodies recapitulated the results with direct GFP fluorescence:DAF-3/GFP is expressed in early embryos; DAF-4/GFP is not. DAF-4/GFP isalso not expressed in membrane surrounding the intestinal lumen, unlikeDAF-3/GFP.

[0205] The combination of the DAF-3 and DAF-4 expression patternssuggests that these genes act in target tissues to transducepheromone-regulated DAF-7 neuroendocrine signals. The early expressionof DAF-3 in embryos is also consistent with a model that DAF-3 actsduring embryonic development, for example, to mediate the development ofneuronal pathways that emit neuroendocrine signals that antagonize DAF-7TGF-β signaling during the L1 stage. However our data indicates thatDAF-3 functions in transducing environmental signals during the L1 andL2 stages. This is supported by the following observations. (1) DAF-7TGF-β signal from ASI neurons occurs during the L1 and L2 stages and isrepressed by dauer-inducing environmental conditions. (2) Expression ofthe DAF-4 type II receptor begins in very late embryogenesis. (3)Expression patterns of DAF-3 and DAF-4 are coincident in most of thetissues remodeled during dauer morphogenesis. For example, the cuticlesecreted by the hypodermis is modified, the pharynx is slimmed, and thelumen of the intestine is less convoluted. In addition, somatic gonaddevelopment is arrested in dauers, and the distal tip cell, in whichDAF-3 is expressed, is an important regulator of that development(Kimble, Developmental Biology 87:286, 1981). In addition, the intestineand hypodermis of dauer larvae contain large fat stores indicative of ametabolic shift to fat storage. The expression of both the DAF-4 TGF-βfamily receptor kinase and the DAF-3 Smad protein in these targettissues is consistent with a model that the DAF-7 neuroendocrine signalfrom the ASI neuron is received directly by these tissues during nondauer development. In addition, the observation that DAF-4 and DAF-3 areexpressed in many of the same cells is consistent with a model thatDAF-4 signaling to downstream Smads (DAF-8 and DAF-14 are likelycandidates) directly regulates DAF-3 gene activity. The TGF-β regulatednuclear localization and transcriptional activation of some Smadproteins suggests that DAF-3 might induce the dauer-specific changes byactivating transcription in target tissues of genes required for dauerformation or repressing transcription of genes necessary for nondauergrowth.

[0206] Smad1 and Smad2 relocalize to become predominantly nuclear whenthe upstream TGF-β signaling pathways are activated (Baker and Harland,Genes and Development 10: 1880, 1996; Hoodless et al., Cell 85:489,1996; Liu et al., Nature 381:620, 1996; Macias-Silva et al., Cell87:1215, 1996). In wild-type, DAF-3/GFP is primarily, although notexclusively, cytoplasmic. DAF-3/GFP subcellular distribution wasexamined in head neurons in the vicinity of ASI (the cell that producesthe DAF-7 signal), as well as in intestinal cells. DAF-3/GFP waspredominantly cytoplasmic in all animals. However, in all animals, dimGFP fluorescence was observed in the nucleus of some of the cells withbright fluoresence, and in approximately twenty-five percent of theanimals, equivalent DAF-3/GFP levels in the nucleus and cytoplasm hasobserved in one or more cells.

[0207] Because DAF-3 is antagonized by the other members of the DAF-7TGF-β pathway, we expect that DAF-3 is active (and perhaps localized tothe nucleus) when these genes are inactive. We therefore observed thesubcellular localization of the full-length DAF-3/GFP fusion protein inthe head neurons, tail neurons, and intestine of dauer-constitutivemutant L1 worms, when DAF-3 gene activity is predicted to be highest. InDAF-1(m402), daf-4(m72), daf-7(m62), daf-8(sa233), and daf-14(m77)mutants, DAF-3/GFP was predominantly cytoplasmic, although, as inwild-type, cells were seen with some GFP in the nucleus. In threedaf-4(m 72) mutant lines, DAF-3/GFP was localized to the nucleus morethan in wild-type lines. When these strains were crossed to wild-type,the increased nuclear localization was seen in both the daf-4 andwild-type segregants. Thus the increased nuclear GFP was a property ofthe array, rather than of daf-4. Even in the neurons nearest to ASI,where the DAF-7 signal should be strongest, no change in DAF-3/GFPsubcellular localization was detected. The DAF-3/GFP fusion protein ispredominantly cytoplasmic in L1 and L2 stages of larvae induced to formdauers by environmental conditions or by mutations in the insulinreceptor pathway gene daf-2, rather than by mutations in the DAF-7signaling pathway mutants (data not shown). The tissue-specificexpression pattern of DAF-3/GFP was unaltered in these mutantbackgrounds (data not shown).

[0208] The finding that DAF-3/GFP subcellular localization is notstrongly responsive to DAF-7 signaling defects or to dauer-inducingenvironmental conditions does not rule out a role for DAF-3 in thenucleus in dauer formation. Even though we detect no change in DAF-3/GFPsubcellular localization, we do detect some DAF-3/GFP in nuclei, and aminor change in nuclear localization or a change in activity due tophosphorylation state may couple DAF-3 to DAF-7 signaling. In fact, thesubcellular localization of Drosophila MAD protein is not detectablyaltered in wild-type when receptor signaling to MAD occurs;relocalization is seen only if the DPP ligand is drasticallyoverexpressed. It is unlikely that a set of undiscovered TGF-β receptorsregulates DAF-3. The C. elegans genome sequence is 90% complete, andthere is only one candidate TGF-β receptor gene other than daf-1 anddaf-4. If this receptor were a positive regulator of DAF-3, mutantswould be expected to, like daf-3 mutants, suppress daf 7 mutants. Thisreceptor acts in a signaling pathway distinct from DAF-3, and it is nota suppressor of daf-7.

[0209] The implication from Smad homology that DAF-3 is active in thenucleus is supported by two additional observations. First, DAF-3/GFP isassociated with chromosomes in intestinal cells during mitosis. Thesecells divide at the end of the L1 stage, and antibody staining withanti-GFP antibodies and anti-α-tubulin antibodies reveals that DAF-3/GFPis found associated with DNA between the spindles during mitosis (FIG.8A). We see DAF-3 GFP co-localized with DAPI from prophase to lateanaphase. DAF-3/GFP was associated with nuclei in prophase by thefollowing criteria. The spindles were present on either side of thenucleus, but the nucleus has not completely broken down. In particular,an indistinct nucleolus was present. DAF-3/GFP continues to co-localizewith DAPI until the chromosomes have separated to the normal distance bywhich nuclei are separated in the intestine, implying continuedassociation until telophase. At this point in mitosis, DAF-3/GFP fadesand becomes undectectable before the nuclei reform the nuclear envelopeand nucleolus. Thus, DAF-3 can, indirectly or directly, bind DNA,consistent with the hypothesis that it is a transcriptional activatorthat acts in the nucleus. DAF-3 is not predicted from its mutantphenotype to have a role in mitosis. It is possible that the brighterGFP on mitotic chromosomes is due to increased access to DNA due to thebreakdown of the nuclear envelope. The second indication of DAF-3function in the nucleus is our examination of a truncated DAF-3/GFPfusion that is missing most of conserved domain II. The truncatedconstruct pGP7 consists of 8 kb of daf-3 fused to GFP. An 8 kb EcoRlfragment from B0217 was cloned into the EcoRl site of pBluescript SK(−).A Pvul/SalI fragment of this subclone was ligated to a Pvul/SalIfragment from the GFP vector pPD95.81. The resulting plasmid contains˜2.5 kb of sequence upstream of the 5′-most exon of daf-3 and codingregion through the first 58 amino acid residues of domain II. Theremaining 175 amino acids of daf-3 and the 3′ noncoding region arereplaced with GFP and the unc-54 3′ end. Three transgenic lines wereisolated, and all had a similar phenotype. This fusion proteininterferes with dauer induction; like a daf-3 loss-of-function mutant,it suppresses mutations in daf-7 (FIG. 7). This truncated protein ispredominantly nuclear, suggesting that it represses dauer formation byacting in the nucleus (FIG. 8B). This result implies that wild-typeDAF-3 also has a function in the nucleus. The full-length DAF-3/GFPconstruct also suppresses mutations in daf-7, as does a full-lengthDAF-3 construct without GFP (FIG. 7). This suppression indicates thatoverexpression of DAF-3 in the cytoplasm has dominant-negative activity,perhaps due to interference with DAF-3 interactions with receptors orcofactors such as other Smads.

[0210] The constitutive nuclear localization of truncated DAF-3/GFPfusion gene missing part of domain II suggests that control of Smadlocalization is complex. A Smad2 construct containing only the conserveddomain II of the protein is constitutively nuclear, leading to thesuggestion that the C-terminus is an effector domain, and the N-terminustethers the protein in the cytoplasm (Baker and Harland, Genes andDevelopment 10:1880, 1996; Hoodless et al., Cell 85:489, 1996; Liu etal., Nature 381:620, 1996; and Macias-Silva et al., Cell 87:1215, 1996).Our construct, in which the N-terminus is intact, is nuclear. Perhapsboth domains provide tethering in the cytoplasm, and any disruptionleads to nuclear entry. Alternatively, entry may be differentlyregulated for DAF-3 and Smad2. Significantly, Smad2, like Smad1 andSmad3 has an SSXS motif at the C terminus (Zhang et al., Nature 383:168,1996; Lagna et al., Nature 383:832, 1996; Savage et al., PNAS 93:790;Baker and Harland, Genes and Development 10:1880, 1996; Hoodless et al.,Cell 85:489, 1996; Liu et al., Nature 381:620, 1996; Macias-Silva etal., Cell 87:1215, 1996; and Graf et al., Cell 85:479, 1996); this motifis a substrate for phosphorylation and required for nuclear localizationof Smad2 (Baker and Harland, Genes and Development 10:1880, 1996;Hoodless et al., Cell 85:489, 1996; Liu et al., Nature 381:620, 1996;and Macias-Silva et al., Cell 87:1215, 1996). DAF-3 has a single serinein the C terminal region, and DPC4 has no serines at this location.

[0211] We propose a model for the TGF-β pathway in dauer formation(FIGS. 9A-B). The DAF-7 TGF-β ligand, which is produced by the ASIsensory neuron in conditions that induce reproductive organ (Ren et al.,Science 274:1389, 1996; Schakwitz et al., Neuron 17:719, 1996), binds tothe DAF-1/DAF-4 receptor kinases on target tissues. These receptorkinases then phosphorylate the Smads DAF-8 and/or DAF-14, analogous tothe phosphorylation and activation of Smad1, Smad2, and Smad3 (Zhang etal., Nature 383:168, 1996; Lagna et al., Nature 383:832, 1996; Savage etal., PNAS 93:790, 1996). We propose that DAF-3 functions like itsclosest homolog, DPC4, which dimerizes with phosphorylated Smad1 andSmad2, even under conditions that do not lead to detectable DPC4phosphorylation (Zhang et al., Nature 383:168, 1996; Lagna et al.,Nature 383:832, 1996; and Savage et al., PNAS 93:790). We suggest thatDAF-3 forms dauer-inducing homodimers in the absence of DAF-7 signaling(FIGS. 9A-B) that are disrupted when DAF-3 heterodimerizes with aphosphorylated DAF-8 and/or DAF-14 (FIG. 9B). Because daf-8 and daf-14are only partially redundant (Riddle et al., Nature 290:668, 1981;Vowels and Thomas, Genetics 130:105, 1992; and Thomas et al., Genetics134:1105, 1993), each is likely to perform a unique function in dauerformation. Thus, DAF-3/DAF-8 dimers are proposed to have differentactivity from DAF-3/DAF-14. Perhaps each activates a subset of genesrequired for dauer formation. The formation of DAF-8/DAF-3 and/orDAF-14/DAF-3 heterodimers antagonizes dauer induction by the DAF-3/DAF-3homodimer. A daf-8(sa233); daf-14(m77); daf-3(mgDf90) triple mutant canform some dauers in dauer-inducing conditions (data not shown); wesuggest that activity of the Daf-2 pathway may induce dauer in thismutant background.

[0212] The dauer genetic pathway represents a neuroendocrine pathway forcontrol of a diapause arrest and its associated shifts in metabolism andrates of senescence (Ren et al., Science 274:1389, 1996; Schackwitz etal., Neuron 17:719, 1996; and Georgi et al., Cell 61:635, 1990).Similarly, activins, members of the TGF-β family, were originallyidentified based on their neuroendocrine regulatory activity, forexample, in regulation of gonadotropin signaling (Vale et al., inPeptide Growth Factors and Their Receptors, Spom and Roberts, Eds.,Springer-Verlag, Heidelberg, 1990). The DAF-7 signal is not the onlysignal that is necessary for reproductive development. Because mutationsin the DAF-7 TGF-β pathway and in the DAF-2 insulin-like signalingpathway cause the same dauer arrest phenotypes, we propose that both theDAF-7 TGF-β signals and the DAF-2 insulin-like signals are necessary forreproductive development. The involvement of an insulin-like signalingpathway in diapause with its associated metabolic shifts is consistentwith metabolic regulation by insulin in vertebrates. Genetic experimentsindicate that these pathways act in parallel (Riddle et al., Nature290:668, 1981; Vowels and Thomas, Genetics 130:105, 1992; and Thomas etal., Genetics 134:1105, 1993). In particular, daf-3 mutants efficientlysuppress daf-7 mutants, but not daf-2 mutants, and daf-16 mutantsefficiently suppress daf-2 mutants, but poorly suppress daf-7 mutants.It is not yet clear whether these two signaling pathways coverage ontarget tissues or in other regulatory (e.g., hormone secreting) cells.However, the expression of the DAF-7 receptor pathway genes and theDAF-16 gene in essentially all target tissues suggests that the TGF-βand insulin pathways act there, and therefore that integration mustoccur there. Thus, we suggest in FIGS. 9A and 9B that the DAF-2 pathwayconverges on DAF-3/DAF-8DAF-1 Smad signaling to regulate metabolic geneexpression in target tissues.

[0213] The integration of insulin-like and TGF-β signals in metaboliccontrol has important implications for the molecular basis of diabetes.For example, these converging pathways for dauer control suggest that inhuman metabolic control both a DAF-7-like signal and insulin may benecessary for full metabolic control. Thus, declines in signaling fromthe human homolog of DAF-7 could underlie the insulin resistanceassociated with Type II diabetes. In fact the dauer pheromone has beenreported to be a fatty acid and to cause down-regulation of DAF-7expression (Ren et al., supra). Thus pheromone regulation of metabolismmay be related to mammalian obesity induced diabetes, and a humanmutation in DAF-7 or its receptors is expected to contribute to adiabetic condition, just like mutations in the insulin receptor. Inaddition if obesity or age or both cause human DAF-7 to decline, e.g.,under high leptin conditions, such a result would explain lateonset/obesity related diabetes.

[0214] Cloning Mammalian DAF Sequences

[0215] Based on our isolation of novel nematode DAF cDNAs, the isolationof mammalian DAF nucleic acid sequences, including human DAF sequences,is made possible using the sequences described herein and standardtechniques. In particular, using all or a portion of a nematode DAFsequence, one may readily design oligonucleotide probes, includingdegenerate oligonucleotide probes (i.e., a mixture of all possiblecoding sequences for a given amino acid sequence). Theseoligonucleotides may be based upon the sequence of either strand of theDNA.

[0216] Exemplary probes or primers for isolating mammalian DAF sequencespreferably correspond to conserved blocks of amino acids, for example,conserved DAF motifs. Exemplary motifs are as follows:

[0217] DAF-2 (tyrosine kinase domain) (SEQ ID NO: 33)

[0218] 1242 KFHEWAAQICDGMAYLESLKFCHRDLAARNCMINRDETVKIGDFGMARDLFYHDYYKPSGKRMMPVRWMSPESLKDGKFDSKSDVWSFGVVLYEMVTLGAQPYIGLSNDEVLNYIGMARKVIKKPEC 1368

[0219] DAF-2 (ligand binding domain) (SEQ ID NO: 34)

[0220] 242 NTTCQKSCAYDRLLPTKEIGPGCDANGDRCHDQCVGGCERVNDATACHACKNVYHKGKCIEKCDAHLYLLLQRRCVTREQCLQLNPVLSNKTVPIKATAGLCSDKCPDGYQINPDDHRECRKCVGKCEIVC 372

[0221] DAF-2 (67 amino acid motif) (SEQ ID NO: 79)

[0222] 1158 AIKINVDDPASTENLNYLMEANIMKNFKTNFIVQLYGVISTVQPAMVVMEMMDLGNLRDYLRSKRED 1224

[0223] DAF-2 (54 amino acid motif) (SEQ ID NO: 80)

[0224] 1362 VIKKPECCENYWYKVMKMCWRYSPRDRPTFLQLVHLLAAEASPEFR DLSFVLTD 1415

[0225] DAF-2 (69 amino acid motif) (SEQ ID NO: 81)

[0226] 404 KQDSGMASELKDIFANIHTITGYLLVRQSSPFISLNMFRNLRRIEAKSLFRNLYAITVFENPNLKKLFD 472

[0227] DAF-2 (52 amino acid motif) (SEQ ID NO: 82)

[0228] 98 FPHLREITGTLLVFETEGLVDLRKIFPNLRVIGGRSLIQHYALIIYRN PDLE 149

[0229] DAF-2 (46 amino acid motif) (SEQ ID NO: 83)

[0230] 149 EIGLDKLSVIRNGGVRIIDNRKLCYTKTIDWKHLITSSINDVVVDN 194

[0231] DAF-2 (36 amino acid motif) (SEQ ID NO: 84)

[0232] 1112 YNADDWELRQDDVVLGQQCGEGSFGKVYLGTGNNVV 1147

[0233] DAF-3 (Smad Domain I) (SEQ ID NO: 35)

[0234] 240 FDQKACESLVKKLKDKKNDLQNLIDVVLSKGTKYTGCITIPRTLDGRLQVHGRKGFPHVVYGKLWRFNEMTKNETRHVDHCKHAFEMKSDMVC VNPYHYEIVI 342

[0235] DAF-3 (Smad Domain I) (SEQ ID NO: 36)

[0236] 690 NRYSLGLEPNPIREPVAFKVRKAIVDGIRFSYKKDGSVWLQNRMKYPVFVTSGYLDEQSGGLKKDKVHKVYGCASIKTF 768

[0237] DAF-3 (79 amino acid motif) (SEQ ID NO: 85)

[0238] 819 DSLAKYCCVRVSFCKGFGEAYPER 842

[0239] DAF-16 (forkhead DNA binding domain) (SEQ ID NO: 37)

[0240] 727 KKTTTRRNAWGNMSYAELITTAIMASPEKRLTLAQVYEWMVQNVPYFRDKGDSNSSAGWKNSIRHNLSLHSRFMRIQNEGAGKSSWWVINPDAKPG MNPRRTRERS 1044

[0241] DAF-16 (103 amino acid motif) (SEQ ID NO: 54)

[0242] 242 KKTTTRRNAWGNMSYAELITTAIMASPEKRLTLAQVYEWMVQNVPYFRDKGDSNSSAGWKNSIRHNLSLHSRFMRIQNEGAGKSSWWVINPDAKPG MNPRRTR 344

[0243] DAF-16 (41 amino acid motif) (SEQ ID NO: 55)

[0244] 137 TFMNTPDDVMMNDDMEPIPRDRCNTWPMRRPQLEPPLNSSP 177

[0245] DAF-16 (109 amino acid motif) (SEQ ID NO: 56)

[0246] 236 DDTVSGKKTTTRRNAWGNMSYAELITTAIMASPEKRLTLAQVYEWMVQNVPYFRDKGDSNSSAGWKNSIRHNLSLHSRFMRIQNEGAGKSSWWVI NPDAKPGMNPRRTR 344

[0247] DAF-16 (98 amino acid motif) (SEQ ID NO: 58)

[0248] 372 KPNPWGEESYSDIIAKALESAPDGRLKLNEIYQWFSDNIPYFGERSSPEEAAGWKNSIRHNLSLHSRFMRIQNEGAGKSSWWVINPDAKPGMNP RRTR 469

[0249] Using such motifs, mammalian DAF-2, DAF-3, and DAF-16 genes maybe isolated from sequence databases (for example, by the use of standardprograms such as Pileup). Alternatively, such sequences may be used todesign degenerate oligonucleotide probes to probe large genomic or cDNAlibraries directly. General methods for designing and preparing suchprobes are provided, for example, in Ausubel et al., Current Protocolsin Molecular Biology, 1996, Wiley & Sons, New York, N.Y.; and Guide toMolecular Cloning Techniques, 1987, S. L. Berger and A. R. Kimmel, eds.,Academic Press, New York. These oligonucleotides are useful for DAF geneisolation, either through their use as probes for hybridizing to DAFcomplementary sequences or as primers for various polymerase chainreaction (PCR) cloning strategies. If a PCR approach is utilized, theprimers are optionally designed to allow cloning of the amplifiedproduct into a suitable vector. PCR is particularly useful for screeningcDNA libraries from rare tissue types.

[0250] Hybridization techniques and procedures are well known to thoseskilled in the art and are described, for example, in Ausubel et al.,supra, and Guide to Molecular Cloning Techniques, supra. If desired, acombination of different oligonucleotide probes may be used for thescreening of the recombinant DNA library. The oligonucleotides are, forexample, labelled with ³²P using methods known in the art, and thedetectably-labelled oligonucleotides are used to probe filter replicasfrom a recombinant DNA library. Recombinant DNA libraries (for example,human cDNA libraries, such as hypothalamus- or pancreas-derived cDNAlibraries, particularly for DAF-2 and DAF-7 cDNAs) may be preparedaccording to methods well known in the art, for example, as described inAusubel et al., supra, or may be obtained from commercial sources.

[0251] For detection or isolation of closely related DAF sequences, highstringency hybridization conditions may be employed; such conditionsinclude hybridization at about 42° C. and about 50% formamide; a firstwash at about 65° C., about 2×SSC, and 1% SDS; followed by a second washat about 65° C. and about 0.1% SDS, 1×SSC. Lower stringency conditionsfor detecting DAF genes having less sequence identity to the nematodeDAF genes described herein include, for example, hybridization at about42° C. in the absence of formamide; a first wash at about 42° C., about6×SSC, and about 1% SDS; and a second wash at about 50° C., about 6×SSC,and about 1% SDS.

[0252] As discussed above, DAF-specific oligonucleotides may also beused as primers in PCR cloning strategies. Such PCR methods are wellknown in the art and are described, for example, in PCR Technology, H.A. Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide toMethods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, andT. J. White, eds., Academic Press, Inc., New York, 1990; and Ausubel etal., supra. Again, sequences corresponding to conserved regions in a DAFsequence (for example, those regions described above) are preferred foruse in isolating mammalian DAF sequences. Such probes may be used toscreen cDNA as well as genomic DNA libraries.

[0253] Sequences obtained are then examined (for example, using thePileup program) to identify those sequences having the highest aminoacid sequence identity to the C. elegans sequence, particularly in orbetween conserved DAF domains (for example, those domains describedabove). In one particular example, the human FKHR and AFX genes are 10³³more closely related to the DAF-16 forkhead domain than the next mostclosely related forkhead domain protein, making FKHR and AFX candidatesfor mammalian DAF-16 genes.

[0254] Following isolation of such candidate genes by sequence homology,the genes are then tested for their ability to functionally complement adaf mutation. This is most readily assayed by transformation of thesequence into a C. elegans strain having an appropriate mutantbackground. Exemplary C. elegans transformation techniques aredescribed, for example, in Mello et al., EMBO J. 10: 3959-3970, 1991,and assays for DAF-2, DAF-3, and DAF-16 polypeptide function aredescribed herein. To be considered useful in the invention, a mammaliansequence need not fully complement a C. elegans defect, but must providea detectable level of functional complementation.

[0255] The DAF, AGE, or AKT gene homologue identified as above, may alsocomplement or alter the metabolic phenotypes of a mammalian cell line.

[0256] For example, addition of DAF-7, TGF-β-like growth factor to aninsulin responsive cell line (e.g., the 3T3-L1 cell line) may accentuateinsulin responsiveness. Similarly genetic transformation of such a cellline with wild type or dominantly activated versions of a DAF, AGE, orAKT gene may alter metabolism. Such perturbations of metabolic controlare stringent tests of candidate genes as DAF, AGE, or AKT homologues.

[0257] In addition, if that mammalian candidate homologue acts in ametabolic control pathway, and is expressed in similar metabolic controltissues (liver, adipose), it is likely to function homologously to DAFproteins from C. elegans. Addition of a wild type or activated DAF, AKT,or AGE protein (for example by VP16 activation of the DAF-3 or DAF-16transcription factors) can confer on cell lines altered metabolicphenotypes. Thus supplying daf, age, or akt gene activity to such a cellline can alter its metabolism. This is one explemplary test ofhomologous DAF function in metabolic control.

[0258] DAF Polypeptide Expression

[0259] In general, DAF polypeptides according to the invention may beproduced by transformation of a suitable host cell with all or part ofDAF-encoding cDNA fragment (e.g., one of the cDNAs described herein orisolated as described above) in a suitable expression vehicle.

[0260] Those skilled in the field of molecular biology will understandthat any of a wide variety of expression systems may be used to providethe recombinant protein. The precise host cell used is not critical tothe invention. The DAF polypeptide may be produced in a prokaryotic host(e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae,insect cells, e.g., Sf9 or Sf21 cells, or mammalian cells, e.g., COS 1,NIH 3T3, or HeLa cells). Such cells are available from a wide range ofsources (e.g., the American Type Culture Collection, Rockland, Md.;also, see, e.g., Ausubel et al., supra). The method of transformation ortransfection and the choice of expression vehicle will depend on thehost system selected. Transformation and transfection methods aredescribed, e.g., in Ausubel et al. (supra); expression vehicles may bechosen from those provided, e.g., in Cloning Vectors: A LaboratoryManual (P. H. Pouwels et al., 1985, Supp. 1987).

[0261] One preferred expression system is the baculovirus system (using,for example, Sf9 cells and the method of Ausubel et al., supra). Anotherbaculovirus system makes use of the vector pBacPAK9 and is availablefrom Clontech (Palo Alto, Calif.).

[0262] Alternatively, an DAF polypeptide is produced in a mammaliansystem, for example, by a stably-transfected mammalian cell line. Anumber of vectors suitable for stable transfection of mammalian cellsare available to the public, e.g., see Pouwels et al. (supra); methodsfor constructing such cell lines are also publicly available, e.g., inAusubel et al. (supra). In one example, cDNA encoding the DAF protein iscloned into an expression vector which includes the dihydrofolatereductase (DHFR) gene. Integration of the plasmid and, therefore, theDAF protein-encoding gene into the host cell chromosome is selected forby inclusion of 0.01-300 μM methotrexate in the cell culture medium (asdescribed in Ausubel et al., supra). This dominant selection may beaccomplished in most cell types. Recombinant protein expression may beincreased by DHFR-mediated amplification of the transfected gene.Methods for selecting cell lines bearing gene amplifications aredescribed in Ausubel et al. (supra); such methods generally involveextended culture in medium containing gradually increasing levels ofmethotrexate. DHFR-containing expression vectors commonly used for thispurpose include pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel etal., supra). Any of the host cells described above or, preferably, aDHFR-deficient CHO cell line (e.g., CHO DHFR⁻ cells, ATCC Accession No.CRL 9096) are among the host cells preferred for DHFR selection of astably-transfected cell line or DHFR-mediated gene amplification.

[0263] In yet other alternative approaches, the DAF polypeptide isproduced in vivo or, preferably, in vitro using a T7 system (see, forexample, Ausubel et al., supra, or other standard techniques).

[0264] Once the recombinant DAF protein is expressed, it is isolated,e.g., using affinity chromatography. In one example, an anti-DAF proteinantibody (e.g., produced as described herein) may be attached to acolumn and used to isolate the DAF protein. Lysis and fractionation ofDAF protein-harboring cells prior to affinity chromatography may beperformed by standard methods (see, e.g., Ausubel et al., supra).

[0265] Once isolated, the recombinant protein can, if desired, befurther purified, e.g., by high performance liquid chromatography (see,e.g., Fisher, Laboratory Techniques In Biochemistry And MolecularBiology, eds., Work and Burdon, Elsevier, 1980).

[0266] Polypeptides of the invention, particularly short DAF polypeptidefragments, may also be produced by chemical synthesis (e.g., by themethods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 ThePierce Chemical Co., Rockford, Ill.).

[0267] These general techniques of polypeptide expression andpurification may also be used to produce and isolate useful DAFfragments or analogs (described herein).

[0268] Anti-DAF Antibodies

[0269] Using any of the DAF polypeptides described herein or isolated asdescribed above, anti-DAF antibodies may be produced by any standardtechnique. In one particular example, a DAF cDNA or cDNA fragmentencoding a conserved DAF domain is fused to GST, and the fusion proteinproduced in E. coli by standard techniques. The fusion protein is thenpurified on a glutathione column, also by standard techniques, and isused to immunize rabbits. The antisera obtained is then itself purifiedon a GST-DAF affinity column, for example, by the method of Finney andRuvkun (Cell 63:895-905, 1990), and is shown to specifically identifyGST-DAF, for example, by Western blotting.

[0270] Polypeptides for antibody production may be produced byrecombinant or peptide synthetic techniques (see, e.g., Solid PhasePeptide Synthesis, supra; Ausubel et al., supra).

[0271] For polyclonal antisera, the peptides may, if desired, be coupledto a carrier protein, such as KLH as described in Ausubel et al, supra.The KLH-peptide is mixed with Freund's adjuvant and injected into guineapigs, rats, or preferably rabbits. Antibodies may be purified by anymethod of peptide antigen affinity chromatography.

[0272] Alternatively, monoclonal antibodies may be prepared using a DAFpolypeptide (or immunogenic fragment or analog) and standard hybridomatechnology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler etal., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol.6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T CellHybridomas, Elsevier, N.Y., 1981; Ausubel et al., supra).

[0273] Once produced, polyclonal or monoclonal antibodies are tested forspecific DAF recognition by Western blot or immunoprecipitation analysis(by the methods described in Ausubel et al., supra). Antibodies whichspecifically recognize a DAF polypeptide described herein are consideredto be useful in the invention. Anti-DAF antibodies, as isolated above,may be used, e.g., in an immunoassay to measure or monitor the level ofDAF polypeptide produced by a mammal or to screen for compounds whichmodulate DAF polypeptide production (for example, in the screensdescribed herein). In one particular example, antibodies to human DAF-7polypeptide are useful for screening blood samples from patients todetermine whether they possess decreased DAF-7 polypeptide levels. Suchantibodies may be used in any immunological assay, for example, an ELISAassay, and a decrease in DAF-7 is taken as an indication of a diabeticcondition, for example, obesity onset Type II diabetes. In anotherparticular example, anti-DAF antibodies are useful for carrying outpedigree analysis. For example, blood samples from individuals may bescreened with anti-DAF-7 antibodies to detect those members of a familywith a predisposition to a diabetic condition. Anti-DAF antibodies mayalso be used to identify cells that express a DAF gene.

[0274] DAF-7 therapy for obesity-onset Type II diabetes

[0275] Our data indicates that DAF-7 represents an endocrine hormone formetabolic control that acts synergistically with insulin. Declines inDAF-7 may be induced by obesity, just as the dauer pheromone, a fattyacid, causes declines in C. elegans DAF-7 production.

[0276] Accordingly, obesity onset Type II diabetes, glucose intolerance,and the associated atherosclerosis may be treated if DAF-7 hormone isinjected intramuscularly or intravenously (FIG. 23).

[0277] In addition, antibodies to human DAF-7 should detect declines inDAF-7 in pre-diabetic, glucose-intolerant, or obesity induced diabetes.Such antibodies will detect DAF-7 levels in blood, just as insulinlevels are detected in metabolic disease.

[0278] DAF-7 therapeutic potential and dosage can be developed in mousemodels of obesity onset diabetes—the db and ob mouse.

[0279] DAF-7 will in injected either intravenously or intramuscularly,in analogy to insulin therapy.

[0280] The decision of which classes of diabetics to treat with DAF-7will come from a combination of blood tests for DAF-7 levels and genetictesting for which daf, age, or akt mutations a particular diabetic,pre-diabetic patient carries.

[0281] Screening Systems for Identifying Therapeutics

[0282] Based on our experimental results, we have developed a number ofscreening procedures for identifying therapeutic compounds (e.g.,anti-diabetic and anti-obesity pharmaceuticals or both) which can beused in human patients. In particular examples, compounds that downregulate daf-3 or daf-16 or their human homologs are considered usefulin the invention. Similarly, compounds that up regulate or activatedaf-1, daf-2, daf-4, daf-7, daf-8, daf-11, daf-14, age-1, and akt (oreach of their corresponding human homologs) are also considered usefulas drugs for the treatment of impaired glucose tolerance conditions,such as diabetes and obesity. In general, the screening methods of theinvention involve screening any number of compounds for therapeuticallyactive agents by employing any number of in vitro or in vivoexperimental systems. Exemplary methods useful for the identification ofsuch compounds are detailed below.

[0283] The methods of the invention simplify the evaluation,identification, and development of active agents for the treatment andprevention of impaired glucose tolerance conditions, such as diabetesand obesity. In general, the screening methods provide a facile meansfor selecting natural product extracts or compounds of interest from alarge population which are further evaluated and condensed to a fewactive and selective materials. Constituents of this pool are thenpurified and evaluated in the methods of the invention to determinetheir anti-diabetic or anti-obesity activities or both.

[0284] Below we describe screening methods for evaluating the efficacyof a compound as anti-diabetic or anti-obesity agents or both. Theseexamples are intended to illustrate, not limit, the scope of the claimedinvention.

[0285] Test Extracts and Compounds

[0286] In general, novel drugs for the treatment of impaired glucosetolerance conditions are identified from large libraries of both naturalproduct or synthetic (or semi-synthetic) extracts or chemical librariesaccording to methods known in the art. Those skilled in the field ofdrug discovery and development will understand that the precise sourceof test extracts or compounds is not critical to the screeningprocedure(s) of the invention. Accordingly, virtually any number ofchemical extracts or compounds can be screened using the exemplarymethods described herein. Examples of such extracts or compoundsinclude, but are not limited to, plant-, fungal-, prokaryotic- oranimal-based extracts, fermentation broths, and synthetic compounds, aswell as modification of existing compounds. Numerous methods are alsoavailable for generating random or directed synthesis (e.g.,semi-synthesis or total synthesis) of any number of chemical compounds,including, but not limited to, saccharide-, lipid-, peptide-, andnucleic acid-based compounds. Synthetic compound libraries arecommercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Inaddition, natural and synthetically produced libraries are produced, ifdesired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

[0287] In addition, those skilled in the art of drug discovery anddevelopment readily understand that methods for dereplication (e.g.,taxonomic dereplication, biological dereplication, and chemicaldereplication, or any combination thereof) or the elimination ofreplicates or repeats of materials already known for their anti-diabeticand anti-obesity activities should be employed whenever possible.

[0288] When a crude extract is found to have anti-diabetic oranti-obesity activities or both, further fractionation of the positivelead extract is necessary to isolate chemical constituents responsiblefor the observed effect. Thus, the goal of the extraction,fractionation, and purification process is the careful characterizationand identification of a chemical entity within the crude extract havinganti-diabetic or anti-obesity activities. The same in vivo and in vitroassays described herein for the detection of activities in mixtures ofcompounds can be used to purify the active component and to testderivatives thereof. Methods of fractionation and purification of suchheterogenous extracts are known in the art. If desired, compounds shownto be useful agents for the treatment of pathogenicity are chemicallymodified according to methods known in the art. Compounds identified asbeing of therapeutic value are subsequently analyzed using any standardanimal model of diabetes or obesity known in the art.

[0289] There now follow examples of high-throughput systems useful forevaluating the efficacy of a molecule or compound in treating (orpreventing) an impaired glucose tolerance condition.

[0290] Nematode Release of Dauer Arrest Bioassays

[0291] To enable mass screening of large quantities of natural products,extracts, or test compounds in an efficient and systematic fashion, C.elegans mutant dauer larvae (e.g., C. elegans containing mutationsdescribed herein, such as C. elegans daf-2 mutant dauer larvae) arecultured in wells of a microtiter plate, facilitating the semiautomationof manipulations and full automation of data collection. As discussedabove, compounds that down regulate DAF-3 or DAF-16 activities or upregulate DAF-1, DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, orAKT activities are considered useful in the invention. Such compoundsare identified by their effect on dauer formation in C. elegans strainscarrying mutations in these genes (as described above).

[0292] In particular examples, nematodes bearing mutations in the DAF-2polypeptide arrest as dauer larvae, never producing progeny. All of themetabolic and growth arrest phenotypes caused by lack of daf-2 aresuppressed by mutations in daf-16. Mutations in the PI 3-kinase, AGE-1,have the same phenotype as lack of daf-2, and such mutations are alsosuppressed by daf-16 mutations. Biochemical analysis of insulinsignaling in mammals supports the view that AGE-1 transduces signalsfrom the DAF-2 receptor by generating a PIP3 signal. Because daf-16mutations suppress lack of daf-2, or age-1 gene activity, it is believedthat PIP3 down regulates or modifies daf-16 gene activity. Thebiochemical overlap between DAF-2/AGE-1 and insulin receptors/PI3-kinase indicates that the human homolog of the C. elegans daf-16 geneacts in the insulin pathway as well. Thus, the C. elegans insulinsignaling pathway yields the surprising result that the animals can livewithout insulin signaling, provided they are mutant in daf-16. Thisanalysis therefore indicates that a compound that inhibits DAF-16activity would reverse the effects of diabetic lesions, e.g., in theproduction or secretion of insulin or in the reception of insulinsignals by target tissues. Such drugs would be expected to beefficacious in the treatment of insulin deficiencies due to pancreatic βcell destruction in Type I diabetes, as well as some Type II diabetesdue to defects in insulin signaling.

[0293] To evaluate the ability of a test compound or an extract todecrease daf-16 gene activity, mutant daf-2 (e1370); daf-16 (mgDf50)animals carrying an integrated human DAF-16 gene are incubated inmicrotiter dishes in the presence of a test compound. This human DAF-16gene supplies all of the DAF-16 activity in the C. elegans strain andthus allows daf-2-induced dauer arrest unless its activity is decreasedby the candidate test compound. If desired, various concentrations ofthe test compound or extract can be inoculated to assess the dosageeffect. Control wells are incubated in the absence of a test compound orextract. Plates are then incubated at 25° C. After an appropriate periodof time, e.g., 2 to 5 days, wells are examined for progeny. The presenceof progeny is taken as an indication that the test compound or extractis effective at inhibiting daf-3 or daf-16 activity, and therefore isconsidered useful in the invention. Any compound that inhibits DAF-16gene activity (or activates upstream signaling in the absence ofreceptor function) will allow reproduction. This is shown schematicallyin FIG. 19.

[0294] Alternatively, a diabetic condition may arise from defects in theDAF-7 TGF-β signaling pathway. Since a decrease in DAF-3 activitybypasses the need for DAF-7 activity in C. elegans metabolic control,drugs that down regulate DAF-3 activity are useful for ameliorating themetabolic defects associated with diabetes. To screen for such drugs,daf-7 (e1372); daf-3 (mg90) nematodes expressing human DAF-3 are exposedto chemicals as described above. In this strain, human DAF-3 suppliesall DAF-3 activity, causing daf-7 induced dauer arrest unless itsactivity is inhibited (FIG. 20). Compounds capable of inhibiting thisactivity are considered useful therapeutics in the invention.

[0295] Finally, in a less complex screen for drugs that inhibit C.elegans daf-3 or daf-16, daf-7 or daf-2 mutants are directly screenedfor compounds that decrease C. elegans daf-3 or daf-16 gene activity.

[0296] In addition, C. elegans worms carrying other daf mutations may beutilized in an assay to obtain additional information on the mode ofaction of the test compound in the insulin or TGF-β signaling pathways.For example, a drug having PIP3 agonist activity would be expected toallow age-1 and daf-2 mutants (but not akt or daf-7 mutants) to notarrest at the dauer stage. Similarly, drugs that inhibit daf-3 areexpected to suppress daf-7 mutants but not daf-2 or age-1 mutants.

[0297] Other Screening Assays

[0298] Other drug screening assays may also be performed using either C.elegans worms or mammalian cell cultures. If desired, such assays mayinclude the use of reporter gene constructs.

[0299] For example, evaluation of the effects of test compounds on dauerformation or reporter gene expression in mutant C. elegans strainsexpressing particular human homologs of the daf, age, or akt genes(i.e., humanized C. elegans) represent useful screening methods.Expression of the human homologs in C. elegans is accomplished accordingto standard methods and, if desired, such genes may be operativelylinked to a gene promoter obtained from C. elegans. Such promotersinclude, without limitation, the C. elegans daf-16, age-1, daf-3, daf-4,and akt gene promoters. For example, the 2.5 kb age-1 promoter can begenerated and isolated by employing standard PCR methods using thefollowing primers: 5′GGAAATATTTTAGGCCAGATGCG3′ (SEQ IS NO: 49) and5′CGGACAGTCCTGAATACACC3′ (SEQ ID NO: 50).

[0300] Additionally, mammalian tissue culture cells expressing C.elegans daf age-1, or akt homologs may be used to evaluate the abilityof a test compound or extract to modulate the insulin or TGF-β signalingpathways. Because the signaling pathways from the ligands, receptors,kinase cascades, and downstream transcription factors are conserved fromman to worm, test compounds or extracts that inhibit or activate theworm signaling proteins should also inhibit or activate their respectivehuman homolog. For example, our identification that DAF-16 is atranscription factor that acts downstream of insulin-like signaling inC. elegans indicates that human DAF-16 transcription reporter genes alsocan be used to identify drugs that inhibit any of the kinases in thesignaling pathway downstream of insulin signaling. For example, the useof DAF-16 and DAF-3 protein binding sites in reporter gene constructsmay be used to monitor insulin signaling. Candidate compounds mimickinginsulin signaling (e.g., PIP3 agonists) are expected to increasereporter gene expression and are considered useful in the invention.

[0301] Reporter Gene Construct

[0302] In one particular example, the invention involves the use of areporter gene that is expressed under the control of a C. elegans genepromoter, e.g., a promoter that includes theTCTCGTTGTTTGCCGTCGGATGTCTGCC (SEQ ID NO: 51) enhancer element, such asthe C. elegans pharyngeal myosin promoter (Okkema and Fire, Development120: 2175-2186, 1994). This enhancer element is known to respond toDAF-3 regulation (i.e., in daf-7 mutants, where daf-3 is active, theelement confers high level expression to reporter genes; whereas in adaf-7; daf-3 mutant (for example, daf-7 (e1372); daf-3), the elementconfers low level expression to reporter genes). Other equivalentenhancer elements may also be used in the invention, e.g., the enhancerelement which is bound by the Xenopus Smad1 and Fast1 forkhead proteins(Nature 383 600-608, 1996). The enhancer element is cloned upstream ofany standard reporter gene, e.g., the luciferase or green fluorescentprotein (GFP) reporter genes. In preferred embodiments, the GFP reportergene is used in C. elegans. In other preferred embodiments, either theGFP or the luciferase reporter genes may used in a mammalian cell basedassay. The reporter gene construct is subsequently introduced into anappropriate host (e.g., C. elegans or a mammalian cell) according to anystandard method known in the art. Analysis of reporter gene activity inthe host organism or cell is determined according to any standardmethod, e.g., those methods described herein. Such reporter gene (andhost cell systems) are useful for screening for drugs that modulateinsulin or DAF-7 metabolic control signaling.

[0303]C. elegans

[0304] In one working example, the above-described reporter geneconstruct is introduced into wild-type C. elegans according to standardmethods known in the art. If the enhancer element is operational, thenit is expected that reporter gene expression is turned on.Alternatively, in daf mutants (e.g., daf-7 or daf-2 mutants, whereinsulin signaling is defective) carrying the above-described reportergene construct, reporter gene activity is turned off.

[0305] Using this on/off distinction, test compounds or extracts areevaluated for the ability to disrupt the signaling pathways describedherein. In one working example, daf-2 mutant worms carrying the reportergene construct are used to assay the expression of the reporter gene.The majority of worms expressing the reporter gene will arrest at thedauer stage because of the daf-2 phenotype. If however the test compoundor extract inhibits DAF-16 activity, then the worms will exhibit adaf-2; daf-16 phenotype (i.e., do not arrest), developing to produceeggs. Such eggs are selected using a bleach treatment and reporter geneexpression in the test compound or extract is assayed according tostandard methods, e.g., worms are examined with an automated fluorometerto reveal the presence of reporter gene expression, e.g., GFP. Candidatecompounds that suppress the daf-2 phenotype or turn on reporter geneexpression, i.e., activate signals in the absence of DAF-2 receptor(e.g., PIP3 mimetics) or inactivate DAF-16, are considered useful in theinvention.

[0306] Analogous screens may also be performed using daf-7 mutants as ameans to identify drugs that inactivate other daf-genes, such as DAF-3,or compounds that activate the DAF-1/DAF-4 receptors. Such screens maybe coupled to reporter screens, for example, using GFP reporter geneswhose expression is under DAF-3 transcriptional control (e.g., the myoIIelement). Drugs identified in such screens are useful as DAF-7 mimetics.Because DAF-7 expression may be down regulated in obesity, such drugsare useful in the treatment of obesity induced Type II diabetes

[0307] In yet another working example, C. elegans DAF-3 and DAF-16 genescan be replaced with a human homolog, (e.g., FKHR for DAF-16), andscreens similar to those described above performed in the nematodesystem. Because drugs may act upstream of the transcription factors, itis useful to replace DAF-1, DAF-4, DAF-8,DAF-14, DAF-2, DAF-3, DAF-16,or AGE-1 with the appropriate human homolog, and to screen the humanizedC. elegans animals. Such screens are useful for identifying compoundshaving activities in humans.

[0308] Mammalian Cells

[0309] Mammalian insulin-responsive cell lines are also useful in thescreening methods of the invention. Here reporter gene constructs (forexample, those described above) are introduced into the cell line toevaluate the ability of a test compound or extract to modulate insulinand TGF-β signaling pathways using a second construct expressing a C.elegans daf, age, or akt gene or their corresponding human homologs.Exemplary cell lines include, but are not limited to, mouse 3T3, L6, andL1 cells (MacDougald et al., Ann. Rev. Biochem. 64: 345-373, 1995)Introduction of the constructs into such cell lines is carried outaccording to standard methods well known in the art.

[0310] To test a compound or extract, it is added to the cell line, andreporter gene expression is monitored. Compounds that induce reportergene expression in the absense of insulin or DAF-7 signaling areconsidered useful in the invention. Such compounds may also turn thecells into adipocytes, as insulin does.

[0311] Compounds identified in mammalian cells may be tested in otherscreening assays described herein, and, in general, test compounds maybe assayed in multiple screens to confirm involvement in insulin orDAF-7 signaling.

[0312] Metabolic control by DAF-7 protein may be tested using any knowncell line, e.g., those described herein.

[0313] In Vitro Screening Methods

[0314] A variety of methods are also available for identifying usefulcompounds in in vitro assays. In one particular example, test compoundsare screened for the ability to activate the phosphorylation of Smadproteins, DAF-8, DAF-14, or DAF-3, by DAF-1 or DAF-4 in vitro. In theseassays, DAF-8, DAF-14, or DAF-3 is preferably tagged with a heterologousprotein domain, for example, the myc epitope tag domain(s) by the methodof Ausubel et al., and are incubated with the C-terminal kinase domainof DAF-1 or DAF-4. Phosphorylation of the Smad proteins is preferablydetected by immunoprecipitation using antibodies specific to the tag,followed by scintillation counting. Test compounds may be screened inhigh throughout microtiter plate assays. A test compound thateffectively stimulates the phosphorylation of DAF-8, DAF-14, or DAF-3 isconsidered useful in the invention. Using these same general assays,compounds that activate the phosphorylation of DAF-16 by AKT or GSK-3may also be identified.

[0315] In another working example, test compounds are screened for theability to inhibit the in vitro association of DAF-16 and the Smadproteins DAF-3, or preferentially activates the association of DAF-16with DAF-8 and DAF-14, DAF-8, or DAF-14, or to inhibit the associationof DAF-3 and DAF-16 with DNA in vitro. These assays are carried out byany standard biochemical methods that test protein-protein binding orprotein-DNA binding. In one particular example, to test for interactionsbetween proteins, each protein is tagged with a different heterologousprotein domain (as described above). Immunoprecipitations are carriedout using an antibody to one tag, and an ELISA assay is carried out onthe immunoprecipitation complex to test for the presence of the secondtag. In addition, if interaction capability is enhanced by a DAF or AKTkinase, this protein is also preferably included in the reactionmixture. Similarly, to test for interactions of these proteins with DNA,antibodies to the tag are utilized in immunoprecipitations, and thepresence of the DNA detected by the presence of the DNA label in theimmunoprecipitation complex. A test compound that effectively inhibitsthe association between these proteins or between DAF-3 and DAF-16 withDNA or both is considered useful in the invention.

[0316] In still another working example, test derivatives of PIP3 arescreened for the ability to increase in vitro AKT activity. This isaccomplished, in general, by combining a labeled PIP3 and an AKTpolypeptide in the presence and absence of the test compound underconditions that allow PIP3:AKT to bind in vitro. Compounds are thenidentified that interfere with the formation of the PIP3:AKT complex.Test compounds that pass this first screen may then be tested forincreased AKT activation in vitro using GSK3 targets, or may be testedin nematodes or mammalian cells (as described above). An increase in AKTkinase activity is taken as an indication of a compound useful forameliorating or delaying an impaired glucose tolerance condition.

[0317] In yet another working example, DAF-3 or DAF-16 may be expressedin a yeast one-hybrid assay for transcriptional activation. Methods forsuch assays are described in Brent and Ptashne (Cell 43:729-736, 1985).A test compound that blocks the ability of DAF-3 or DAF-16 or both toactivate (or repress) transcription in this system is considered usefulin the invention.

[0318] In a final working example, compounds may be screened for theirability to inhibit an interaction between any of DAF-3, DAF-8, andDAF-14, or between DAF-3 and DAF-16. These in vivo assays may be carriedout by any “two-hybrid” or “interaction trap” method (for example, byusing the methods described by Vijaychander et al (Biotechniques 20:564-568)).

[0319] Modulatory Compounds

[0320] Our experimental results facilitate the isolation of compoundsuseful in the treatment of impaired glucose tolerance diseases that areantagonists or agonists of the insulin or TGF-β signaling pathwaysidentified in C. elegans and described above. Exemplary methods for theisolation of such compounds now follow.

[0321] Antagonists

[0322] As discussed above, useful therapeutic compounds include thosewhich down regulate the expression or activity of DAF-3 or DAF-16. Toisolate such compounds, DAF-3 or DAF-16 expression is measured followingthe addition of candidate antagonist molecules to a culture medium ofDAF-3 or DAF-16-expressing cells. Alternatively, the candidateantagonists may be directly administered to animals (for example,nematodes or mice) and used to screen for their effects on DAF-3 orDAF-16 expression.

[0323] DAF-3 or DAF-16 expression is measured, for example, by standardNorthern blot analysis (Ausubel et al., supra) using a DAF-3 or DAF-16nucleic acid sequence (or fragment thereof) as a hybridization probe.The level of DAF-3 or DAF-16 expression in the presence of the candidatemolecule is compared to the level measured for the same cells, in thesame culture medium, or in a parallel set of test animals, but in theabsence of the candidate molecule. Preferred modulators foranti-diabetic or anti-obesity purposes are those which cause a decreasein DAF-3 or DAF-16 expression.

[0324] Alternatively, the effect of candidate modulators on expressionor activity may be measured at the level of DAF-3 or DAF-16 proteinproduction using the same general approach in combination with standardimmunological detection techniques, such as Western blotting orimmunoprecipitation with a DAF-3 or DAF-16-specific antibody (forexample, the DAF-3 or DAF-16 antibodies described herein). Again, usefulanti-diabetic or anti-obesity therapeutic modulators are identified asthose which produce a decrease in DAF-3 or DAF-16 polypeptideproduction. Antagonists may also affect DAF-3 or DAF-16 activity withoutany effect on expression level. For example, the identification ofkinase cascades upstream of DAF-3 and DAF-16 (as described herein)suggest that the phosphorylation state of these polypeptides iscorrelated with activity. Phosphorylation state may be monitored bystandard Western blotting using antibodies specific for phosphorylatedamino acids. In addition, because DAF-3 and DAF-16 are transcriptionfactors, reporter genes bearing operably linked DAF-3 or DAF-16 bindingsites (for example, the myoII enhancer element) may be used to directlymonitor the effects of antagonists on DAF-3 or DAF-16 gene activity.

[0325] Candidate modulators may be purified (or substantially purified)molecules or may be one component of a mixture of compounds (e.g., anextract or supernatant obtained from cells). In a mixed compound assay,DAF-3 or DAF-16 expression is tested against progressively smallersubsets of the candidate compound pool (e.g., produced by standardpurification techniques, e.g., HPLC or FPLC; Ausubel et al., supra)until a single compound or minimal compound mixture is demonstrated tomodulate DAF-3 or DAF-16 expression.

[0326] Candidate DAF-3 or DAF-16 antagonists include peptide as well asnon-peptide molecules (e.g., peptide or non-peptide molecules found,e.g., in a cell extract, mammalian serum, or growth medium on whichmammalian cells have been cultured).

[0327] Antagonists found to be effective at the level of cellular DAF-3or DAF-16 expression or activity may be confirmed as useful in animalmodels (for example, nematodes or mice). For example, the compound mayameliorate the glucose intolerance and diabetic symptoms of mouse modelsfor Type II diabetes (e.g., a db mouse model), mouse models for Type Idiabetes, or models of streptozocin-induced β cell destruction.

[0328] A molecule which promotes a decrease in DAF-3 or DAF-16expression or DAF-3 or DAF-16 activity is considered particularly usefulin the invention; such a molecule may be used, for example, as atherapeutic to decrease the level or activity of native, cellular DAF-3or DAF-16 and thereby treat a glucose intolerance condition in an animal(for example, a human).

[0329] If desired, treatment with an antagonist of the invention may becombined with any other anti-diabetic or anti-obesity therapies.

[0330] Agonists

[0331] Also as discussed above, useful therapeutic compounds are thosewhich up regulate the expression or activity of DAF-1, DAF-2, DAF-4,DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT. To isolate such compounds,expression of these genes is measured following the addition ofcandidate agonist molecules to a culture medium of DAF-1, DAF-2, DAF-4,DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT-expressing cells.Alternatively, the candidate agonists may be directly administered toanimals (for example, nematodes or mice) and used to screen for effectson DAF-1, DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKTexpression.

[0332] DAF-1, DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, orAKT-expression is measured, for example, by standard Northern blotanalysis (Ausubel et al., supra) using all or a portion of one of thesegenes as a hybridization probe. The level of DAF-1, DAF-2, DAF-4, DAF-7,DAF-8, DAF-11, DAF-14, AGE-1, or AKT expression in the presence of thecandidate molecule is compared to the level measured for the same cells,in the same culture medium, or in a parallel set of test animals, but inthe absence of the candidate molecule. Preferred modulators foranti-diabetic or anti-obesity purposes are those which cause an increasein DAF-1, DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKTexpression.

[0333] Alternatively, the effect of candidate modulators on expressionmay be measured at the level of DAF-1, DAF-2, DAF-4, DAF-7, DAF-8,DAF-11, DAF-14, AGE-1, or AKT protein production using the same generalapproach in combination with standard immunological detectiontechniques, such as Western blotting or immunoprecipitation with anappropriate antibody. Again, the phosphorylation state of thesepolypeptides is indicative of DAF activity and may be measured onWestern blots. Useful anti-diabetic or anti-obesity modulators areidentified as those which produce an increase in DAF-1, DAF-2, DAF-4,DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT polypeptide production.

[0334] Candidate modulators may be purified (or substantially purified)molecules or may be one component of a mixture of compounds (e.g., anextract or supernatant obtained from cells). In a mixed compound assay,DAF-1, DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKTexpression is tested against progressively smaller subsets of thecandidate compound pool (e.g., produced by standard purificationtechniques, e.g., HPLC or FPLC; Ausubel et al., supra) until a singlecompound or minimal compound mixture is demonstrated to modulate DAF-1,DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT expression.

[0335] Alternatively, or in addition, candidate compounds may bescreened for those which agonize native or recombinant DAF-1, DAF-2,DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT activities. In oneparticular example, DAF-1 and DAF-4 phosphorylation of DAF-8 and DAF-14,or AKT phosphorylation of DAF-16, may be activated by agonists.

[0336] Candidate DAF-1, DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14,AGE-1, or AKT agonists include peptide as well as non-peptide molecules(e.g., peptide or non-peptide molecules found, e.g., in a cell extract,mammalian serum, or growth medium on which mammalian cells have beencultured).

[0337] Agonists found to be effective at the level of cellular DAF-1,DAF-2, DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT expression oractivity may be confirmed as useful in animal models (for example,nematodes or mice).

[0338] A molecule which promotes an increase in DAF-1, DAF-2, DAF-4,DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT expression or DAF-1, DAF-2,DAF-4, DAF-7, DAF-8, DAF-11, DAF-14, AGE-1, or AKT activities isconsidered particularly useful in the invention; such a molecule may beused, for example, as a therapeutic to increase the level or activity ofthese native, cellular genes and thereby treat a glucose intolerancecondition.

[0339] If desired, treatment with an DAF-1, DAF-2, DAF-4, DAF-7, DAF-8,DAF-11, DAF-14, AGE-1, or AKT agonist may be combined with any otheranti-diabetic or anti-obesity therapies.

[0340] Animal Model Systems

[0341] Compounds identified as having activity in any of theabove-described assays are subsequently screened in any number ofavailable diabetic or obesity animal model systems, including, but notlimited to ob (Coleman, Dibetologia 14: 141-148, 1978; Chua et al.,Science 271: 994-996, 1996; Vaisse et al., Nature Genet. 14:95-100,1996), db (Chen et al., Cell 84: 491-495, 1996), agouti mice, or fattyrats (Takaga et al. Biochem. Biophys. Res. Comm. 225: 75-83, 1996). Testcompounds are administered to these animals according to standardmethods. Additionally, test compounds may be tested in mice bearingknockout mutations in the insulin receptor, IGF-1 receptor (e.g., Liu etal., Cell 75:59-72, 1993), IR-related receptor, DAF-7 homolog, or any ofthe daf (FKHR, AFX) genes described herein. Compounds can also be testedusing any standard mouse or rat model of Type I diabetes, e.g., astreptozin ablated pancreas model.

[0342] In one particular example, the invention involves theadministration of DAF-7 or its homolog as a method for treating diabetesor obesity. Evaluation of the effectiveness of such a compound isaccomplished using any standard animal model, for example, the animaldiabetic model systems described above. Because these mouse diabeticmodels are also associated with obesity, they provide preferred modelsfor human obesity associated Type II diabetes as well. Such diabetic orobese mice are administered C. elegans or human DAF-7 according tostandard methods well known in the art. Treated and untreated controlsare then monitored for the ability of the compound to ameliorate thesymptoms of the disease, e.g., by monitoring blood glucose,ketoacidosis, and atherosclerosis. Normalization of blood glucose andinsulin levels is taken as an indication that the compound is effectiveat treating a glucose intolerance condition.

[0343] Therapy

[0344] Compounds of the invention, including but not limited to, DAF-7and its homologs, and any antagonist or agonist therapeutic agentidentified using any of the methods disclosed herein, may beadministered with a pharmaceutically-acceptable diluent, carrier, orexcipient, in unit dosage form. Conventional pharmaceutical practice maybe employed to provide suitable formulations or compositions toadminister such compositions to patients. Although intravenousadministration is preferred, any appropriate route of administration maybe employed, for example, parenteral, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, ororal administration. Therapeutic formulations may be in the form ofliquid solutions or suspensions; for oral administration, formulationsmay be in the form of tablets or capsules; and for intranasalformulations, in the form of powders, nasal drops, or aerosols.

[0345] Methods well known in the art for making formulations are foundin, for example, “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for antagonists or agonists of the invention includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

[0346] DAF polypeptides are administered at any appropriateconcentration, for example, for DAF-7, at a concentration of around 10nM.

[0347] Pedigree Analysis and Genetic Testing

[0348] The discovery described herein that DAF polypeptides are involvedin glucose metabolism enables assays for genetic testing to identifythose individuals with predispositions toward the development of glucoseintolerance conditions, such as diabetes or obesity, by determining thepresence of a mutation found in a human gene having identity to any ofthe C. elegans daf-1, daf-2, daf-3, daf-4, daf-7, daf-8, daf-11, daf-14,daf-16, age-1, or akt genes. In one embodiment, the development of thistesting method requires that the individual be a member of a family thathas multiple affected and unaffected members carrying one mutation fromthe list of above-listed genes. Those skilled in the art will understandthat a diabetic or obesity phenotype may be produced by daf, age, or aktmutations found on different chromosomes, and that low resolutiongenetic mapping of the diabetic condition in single family pedigreeswill be sufficient to favor some daf, age, or akt genes over others ascausing the glucose intolerance condition in a particular pedigree. Inone particular example, mutations associated with glucose intolerancemay be found in different genes in, for example, the DAF-7 signalingpathway in each pedigree. In addition, because mutations in a commonpathway can show complex genetic interactions, multiple DAF mutationsmay segregate in single pedigress. These mutations can behaverecessively in some genetic backgrounds and dominantly in others.

[0349] Those skilled in the art further understand that, to determinedisease linkage with a chromosomal marker, it may be necessary toevaluate the association of inheritance patterns of several differentchromosomal markers (for example, from the collection of highlypolymorphic mapped DNA allelic variants) in the genomic DNAs of familymembers and of the clinically affected individuals. Methods commonlyused in determining segregation patterns of human genetic diseases arewell known in the art. In addition, methods are known in the art fordetermining whether individuals in a family are useful for providinginformation to determine co-segregation of an allele with a glucoseintolerance trait.

[0350] Here, fragments of genomic DNA (e.g., RFLP fragments) areprepared from each of the available members of the family, and eachdistinctive DNA allelic variant of the polymorphic chromosome markerwithin the family is evaluated to determine which polymorphisms (i.e.,chromosomal region) is linked with the glucose intolerance phenotypewithin a particular family. It is preferred that the parents of themarker individual be heterozyous for a DNA allelic variant so that thesegregation pattern of the DNA allelic variant linked with the diabeticor obese phenotype in the marker can be recognized. The inheritance ofthe diabetic phenotype can be judged to be dominant or recessive,depending on the pattern of inheritance. Most diabetes is dominantlyinherited, and therefore inbred pedigrees are generally not necessary inthe etiology of the diabetic condition.

[0351] With respect to Type II diabetes, the highest rate of this kindof diabetes in the world is found in American Indians of the Pima tribe.Such families are useful for mapping one particular cause of diabetes,but, in general, diabetes is caused by mutations in a variety of genes,including daf genes. Thus, by testing for low resolution linkage to acandidate daf, age, or akt mutation, and then by sequencing theparticular linked daf gene in affected and unaffected individuals, aparticular daf mutation can be associated with a particular diabeticpedigree.

[0352] Human DAF homologues are mapped to chromosome regions usingstandard methods. For example, the probable DAF-16 homologue FKHR islocated on chromosome 13, and AFX is located on the X chromosome. Anydaf, akt, or age genes mapping to the approximate chromosomal regionsassociated with diabetes or glucose intolerance are sequenced fromaffected and unaffected individuals. Preferably, at least two genes perpedigree of 5-20 affected (and unaffected controls) are sequenced. Thedaf genomic regions are PCR amplified and compared between affected andunaffected DNA samples. Mutations detected in affected individuals areexpected to (but need not) map to conserved domains of the DAF genes.Because it is known that not all carriers of known diabetes-inducingmutations show metabolic defects, we expect that some non-diabeticnon-glucose intolerant family members will carry the same daf mutationas affected family members. For this reason, a correlation of affectedfamily members with a daf mutation is more important than a correlationof nonaffected with no mutation. Those skilled in the art will know thatphenotypic classification of affected and unaffected individuals cangreatly enhance the power of this genetic analysis (Nature Genet. 11:241-247, 1995). In addition, other mutations in the same daf gene areexpected in some but not all diabetic pedigrees. For dominant diabeticinheritance, the affected individuals carry a daf, age, or akt mutationas well as a normal allele. For recessive diabetic inheritance,individuals carry two daf mutations that may be identical or twoindependent mutations in the same gene. In addition, some diabeticindividuals may carry mutations in more than one daf, age, or akt gene(so called non-allelic non-complementation).

[0353] It is routine in the art of genetic counseling to determine riskfactors given the presence of a closely linked molecular genetic markerin the genomic DNA of the individual and when combined with theadditional understanding provided by the pedigree of the individual inthe family. For example, a risk factor may be calculated for anindividual in an age, akt, or daf chromosome family in a manner similarto those described for assessing the risk of other commonly knowngenetic diseases that are known to run in families, e.g., Huntington'sdisease and cystic fibrosis.

[0354] Once mutations in daf, akt, or age genes are associated withdiabetes in a pedigree analysis, diagnostic PCR sequencing of these dafgenes can be used to diagnose glucose intolerant, prediabetic, diabetic,obesity, and atherosclerotic conditions. Preferably, the daf, akt, orage gene regions are PCR amplified from patients and mutations detectedin the daf genes using standard DNA sequencing or oligonucleotidehybridization techniques. The use of such gene sequences or specificantibody probes to the products of these sequences provide valuablediagnostics, particularly in view of the likelihood there exist twoclasses of type II diabetics: those with defects in the TGF-β signalinggenes, and those with defects in insulin signaling genes. Such genetictests will influence whether drugs that affect DAF-7 TGF-β or DAF-2insulin like signals are prescribed.

[0355] To carry out the above analysis (as well as the other screening,diagnostic, and therapeutic methods described herein), mammalianhomologs corresponding to the C. elegans daf-1, age-1, daf-4, daf-8, anddaf-7 genes are isolated as described above for daf-2, daf-3, anddaf-16. Again, standard hybridization or PCR cloning strategies areemployed, preferably utilizing conserved DAF, AGE, or AKT motifs forprobe design followed by comparison of less conserved sequences flankingthese motifs. Exemplary motifs for these genes are as follows:

[0356] DAF-1 (139 amino acid motif) (SEQ ID NO: 13)

[0357] 274 TSGSGMGPTTLHKLTIGGQIRLTGRVGSGRFGNVSRGDYRGEAVAVKVFNALDEPAFHKETEIFETRMLRHPNVLRYIGSDRVDTGFVTELWLVTEYHPSGSLHDFLLENTVNIETYYNLMRSTASGLAFLHNQIGGSK 412

[0358] DAF-1 (62 amino acid motif) (SEQ ID NO: 14)

[0359] 450 EDAASDIIANENYKCGTVRYLAPEILNSTMQFTVFESYQCADVYSFSLVMWETLCRCEDGDV 511

[0360] DAF-1 (31 amino acid motif) (SEQ ID NO: 15)

[0361] 416 KPAMAHRDIKSKNIMVKNDLTCAIGDLGLSL 466

[0362] DAF-1 (72 amino acid motif) (SEQ ID NO: 16)

[0363] 520 IPYIEWTDRDPQDAQMFDVVCTRRLRPTENPLWKDHPEMKHIMEIIKT CWNGNPSARFTSYICRKRMDERQQ 591

[0364] AGE-1 (150 amino acid motif) (SEQ ID NO: 17)

[0365] 991 YFESVDRFLYSCVGYSVATYIMGIKDRHSDNLMLTEDGKYVHIDFGHILGHGKTKLGIQRDRQPFILTEHFMTVIRSGKSVDGNSHELQKFKTLCVEAYEVMWNNRDLFVSLFTLMLGMELPELSTKADLDHLKKTLFCNGESKEEAR KF 1140

[0366] AGE-1 (113 amino acid motif) (SEQ ID NO: 18)

[0367] 826 SPLDPVYKLGEMIIDKAIVLGSAKRPLMLHWKNKNPKSDLHLPFCAMIFKNGDDLRQDMLVLQVLEVMDNIWKAANIDCCLNPYAVLPMGEMIGIIE VVPNCKTIFEIQVGTG 938

[0368] AGE-1 (106 amino acid motif) (SEQ ID NO: 19)

[0369] 642 LAFVWTDRENFSELYVMLEKWKPPSVAAALTLLGKRCTDRVIRKFAVEKLNEQLSPVTFHLFILPLIQALKYEPRAQSEVGMMLLTRALCDYRIGHRLF WLLRAEI 747

[0370] AGE-1 (60 amino acid motif) (SEQ ID NO: 38)

[0371] 91 EIKLSDFKHQLFELIAPMKWGTYSVKPQDYVFRQLNNFGEIEVIFND DQPLSKLELHGTF150

[0372] AKT (121 amino acid motif) (SEQ ID NO: 60)

[0373] 33685 QVLDDHDYGRCVDWWGVGVVMYEMMCGRLPFYSKDHNKLFELIMAGDLRFPSKLSQEARTLLTGLLVKDPTQRLGGGPEDALEICRADFFRTVDWEATYRKEIEPPYKPNVQSETDTSYFD 34047

[0374] AKT (66 amino acid motif) (SEQ ID NO: 61)

[0375] 32314 TMEDFDFLKVLGKGTFGKVILCKEKRTQKLYAIKILKKDVIIAREEVAHTLTENRVLQRCKHPFLT 32511

[0376] AKT (45 amino acid motif) (SEQ ID NO: 62)

[0377] 33509 KLENLLLDKDGHIKIADFGLCKEEISFGDKTSTFCGTPEYL APEV 33643

[0378] AKT (57 amino acid motif) (SEQ ID NO: 63)

[0379] 32667 YFQELKYSFQEQHYLCFVMQFANGGELFTHVRKCGTFSEPRARFY GAEIVLALGYLH32837

[0380] AKT (59 amino acid motif) (SEQ ID NO: 64)

[0381] 31846 STFAIFYFQTMLFEKPRPNMFMVRCLQWTTVIERTFYAESAEVRQRWIHAIESISKKYK 32022

[0382] AKT (33 amino acid motif) (SEQ ID NO: 65)

[0383] 33156 LQELKYSFQTNDRLCFVMEFAIGGDLYYHLNRE 33254

[0384] AKT (21 amino acid motif) (SEQ ID NO: 66)

[0385] 30836 VVIEGWLHKKGEHIRNWRPRF 30898

[0386] AKT (26 amino acid motif) (SEQ ID NO: 67)

[0387] 33276 FSEPRARFYGSEIVLALGYLHANSIV 33353

[0388] DAF-4 (139 amino acid motif) (SEQ ID NO: 20)

[0389] 380 EYWIVTEFHERLSLYELLKNNVISITSANRIIMSMIDGLQFLHDDRPYFFGHPKKPIIHRDIKSKNILVKSDMTTCIADFGLARIYSYDIEQSDLLGQVGTKRYMSPEMLEGATEFTPTAFKAMDVYSMGLVMWEVISR 518

[0390] DAF-4 (61 amino acid motif) (SEQ ID NO: 21)

[0391] 537 IGFDPTIGRMRNYVVSKKERPQWRDEIIKHEYMSLLKKVTEEMWDPEACARITAGCAFARV 597

[0392] DAF-4 (20 amino acid motif) (SEQ ID NO: 22)

[0393] 305 PITDFQLISKGRFGKVFKAQ 324

[0394] DAF-8 (163 amino acid motif) (SEQ ID NO: 23)

[0395] 382 TDSETRSRFSLGWYNNPNRSPQTAEVRGLIGKGVRFYLLAGEVYVENLCNIPVFVQSIGANMKNGFQLNTVSKLPPTGTMKVFDMRLFSKQLRTAAEKTYQDVYCLSRMCTVRVSFCKGWGEHYRRSTVLRSPVWFQAHLNNPMHW VDSVLTCMGAPPRICSS 544

[0396] DAF-8 (44 amino acid motif) (SEQ ID NO: 24)

[0397] 91 RAFRFPVIRYESQVKSILTCRHAFNSHSRNVCLNPYHYRWVELP 134

[0398] DAF-8 (38 amino acid motif) (SEQ ID NO: 25)

[0399] 341 VEYEESPSWLKLIYYEEGTMIGEKADVEGHHCLIDGFT 378

[0400] DAF-14 (39 amino acid motif) (SEQ ID NO: 68)

[0401] 9709 IRVSFCKGFGETYSRLKVVNLPCWIEIILHEPADEYDTV 9825

[0402] DAF-14 (45 amino acid motif) (SEQ ID NO: 69)

[0403] 9409 SRNSKSSQIRNTVGAGIQLAYENGELWLTVLTDQIVFVQCPFLNQ 9543

[0404] DAF-14 (29 amino acid motif) (SEQ ID NO: 70)

[0405] 9160 NEMLDPEPKYPKEEKPWCTIFYYELTVRV 9246

[0406] DAF-14 (29 amino acid motif) (SEQ ID NO: 71)

[0407] 9307 QLGKAFEAKVPTITIDGATGASDECRMSL 9393

[0408] DAF-12 (105 amino acid motif) (SEQ ID NO: 72)

[0409] 103 SPDDGLLDSSEESRRRQKTCRVCGDHATGYNFNVITCESCKAFFRRNALRPKEFKCPYSEDCEINSVSRRFCQKCRLRKCFTVGMKKEWILNEEQLR RRKNSRLN 207

[0410] DAF-12 (89 amino acid motif) (SEQ ID NO: 73)

[0411] 109 LDSSEESRRRQKTCRVCGDHATGYNFNVITCESCKAFFRRNALRPKEFKCPYSEDCEINSVSRRFCQKCRLRKCFTVGMKKEWILNEEQ 197

[0412] DAF-12 (73 amino acid motif) (SEQ ID NO: 74)

[0413] 551 DIMNIMDVTMRRFVKVAKGVPAFREVSQEGKFSLLKGGMIEMLTVRGVTRYDASTNSFKTPTIKGQNVSVNVD 623

[0414] DAF-11 (112 amino acid motif) (SEQ ID NO: 75)

[0415] 708 SGSLVDLMIKNLTAYTQGLNETVKNRTAELEKEQEKGDQLLMELLPKSVANDLKNGIAVDPKVYENATILYSDIVGFTSLCSQSQPMEVVTLLSGM YQRFDLIISQQGGYKV 819

[0416] DAF-11 (107 amino acid motif) (SEQ ID NO: 76)

[0417] 825 METIGDAYCVAAGLPVVMEKDHVKSICMIALLQRDCLHHFEIPHRPGTFLNCRWGFNSGPVFAGVIGQKAPRYACFGEAVILASKMES SGVEDRIQ MTLASQQLLEE 931

[0418] DAF-11 (43 amino acid motif) (SEQ ID NO: 77)

[0419] 520 DILKGLEYIHASAIDFHGNLTLHNCMLDSHWIVKLSGFGVNRL 562

[0420] DAF-11 (15 amino acid motif) (SEQ ID NO: 78)

[0421] 618 DMYSFGVILHEIILK 632

[0422] DAF-7 (60 amino acid motif) (SEQ ID NO: 26)

[0423] 290 NLAETGHSKIMRAAHKVSNPEIGYCCHPTEYDYIKLIYVNRDGRVSIA NVNGMIAKKCGC349

[0424] DAF-7 (20 amino acid motif) (SEQ ID NO: 27)

[0425] 265 DWIVAPPRYNAYMCRGDCHY 284

[0426] DAF-7 (43 amino acid motif) (SEQ ID NO: 28)

[0427] 240 VCNAEAQSKGCCLYDLEIEFEKIGWDWIVAPPRYNAYMCRGDC 282

[0428] DAF-7 (70 amino acid motif) (SEQ ID NO: 29)

[0429] 281 DCHYNAHHFNLAETGHSKIMRAAHKVSNPEIGYCCHPTEYDYIKLIYV NRDGRVSIANVNGMIAKKCGCS 350

[0430] DAF-7 (35 amino acid motif) (SEQ ID NO: 30)

[0431] 250 CCLYDLEIEFEKIGWDWIVAPPRYNAYMCRGDCHY 284

[0432] DAF-7 (13 amino acid motif)(SEQ ID NO: 51) GWDWIVAPPRYNA

[0433] In one particular example, mammalian DAF-7 may be identifiedusing the sub-domain amino acids 314-323. Exemplary degenerateoligonucleotides designed to PCR amplify this domain or hybridize (forexample, as described in Burglin et al., (Nature 341:239-243, 1989) areas follows:

[0434] aa 263 oligo: GGNTGGGAYTRNRTNRTNGCNCC (23-mer, 16,000-folddegeneracy) (SEQ ID NO: 31)

[0435] aa 314 oligo: TGYTGYNNNCCNACNGAR (18-mer, 8000-fold degeneracy)(SEQ ID NO: 32).

[0436] The DNA sequence between the oligonucleotide probes isdetermined, and those sequences having the highest degree of homologyare selected. Once isolated, these sequences are then tested in a C.elegans daf-7 mutant or mouse model as described above for the abilityto functionally complement the mutation or ameliorate the glucoseintolerance phenotype.

OTHER EMBODIMENTS

[0437] In other embodiments, the invention includes any protein whichpossesses the requisite level of amino acid sequence identity (asdefined herein) to DAF-2, DAF-3, or a DAF-16 sequence; such homologsinclude other substantially pure naturally-occurring mammalian DAFpolypeptides (for example, human DAF polypeptides) as well as allelicvariants; natural mutants; induced mutants; proteins encoded by DNA thathybridizes to the DAF DNA sequence or degenerate conserved domains ofDAF proteins (e.g., those described herein) under high stringencyconditions; and proteins specifically bound by antisera directed to aDAF-2, DAF-3, or DAF-16 polypeptide.

[0438] The invention further includes analogs of any naturally-occurringDAF-2, DAF-3, or DAF-16 polypeptides. Analogs can differ from thenaturally-occurring protein by amino acid sequence differences which donot destroy function, by post-translational modifications, or by both.Modifications include in vivo and in vitro chemical derivatization ofpolypeptides, e.g., acetylation, carboxylation, phosphorylation, orglycosylation; such modifications may occur during polypeptide synthesisor processing or following treatment with isolated modifying enzymes.Analogs can also differ from the naturally-occurring DAF polypeptide byalterations in primary sequence. These include genetic variants, bothnatural and induced (for example, resulting from random mutagenesis byirradiation or exposure to ethanemethylsulfate or by site-specificmutagenesis as described in Sambrook, Fritsch and Maniatis, MolecularCloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel etal., supra). Also included are cyclized peptides, molecules, and analogswhich contain residues other than L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., β or γ aminoacids.

[0439] In addition to full-length polypeptides, the invention alsoincludes DAF-2, DAF-3, and DAF-16 polypeptide fragments. As used herein,the term “fragment,” means at least 20 contiguous amino acids,preferably at least 30 contiguous amino acids, more preferably at least50 contiguous amino acids, and most preferably at least 60 to 80 or morecontiguous amino acids. Fragments of such DAF polypeptides can begenerated by methods known to those skilled in the art or may resultfrom normal protein processing (e.g., removal of amino acids from thenascent polypeptide that are not required for biological activity orremoval of amino acids by alternative mRNA splicing or alternativeprotein processing events).

[0440] For certain purposes, all or a portion of the DAF-2, DAF-3, orDAF-16 polypeptide sequence may be fused to another protein (forexample, by recombinant means). In one example, the DAF polypeptide maybe fused to the green fluorescent protein, GFP (Chalfie et al., Science263:802-805, 1994). Such a fusion protein is useful, for example, formonitoring the expression level of the DAF polypeptide in vivo (forexample, by fluorescence microscopy) following treatment with candidateor known DAF agonists or antagonists.

[0441] The methods of the invention may be used to diagnose or treat anycondition related to glucose intolerance or obesity in any mammal, forexample, humans, domestic pets, or livestock. Where a non-human mammalis diagnosed or treated, the DAF polypeptide, nucleic acid, or antibodyemployed is preferably specific for that species.

[0442] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0443] Other embodiments are within the following claims.

What is claimed is:
 1. A method for identifying a modulatory compoundthat is capable of decreasing the expression or activity of a daf-16gene, said method comprising: (a) providing a nematode, isolatednematode cell, or isolated mammalian cell expressing a daf-16 gene; and(b) contacting said nematode, isolated nematode cell, or isolatedmammalian cell with a candidate compound, a decrease in daf-16expression or activity following contact of said nematode, said isolatednematode cell, or said isolated mammalian cell with said candidatecompound identifying a modulatory compound.
 2. The method of claim 1,wherein said compound is a candidate compound for ameliorating ordelaying an impaired glucose tolerance condition, atherosclerosis, orobesity.
 3. The method of claim 1, wherein said nematode is C. elegans.4. The method of claim 1, wherein said daf-16 gene is a nematode daf-16gene.
 5. A method for the identification of a compound that is acandidate compound for ameliorating or delaying an impaired glucosetolerance condition, said method comprising the steps of: (a) providinga daf-2, daf-16 mutant nematode; (b) expressing in the cells of saidnematode a mammalian AFX polypeptide, whereby said nematode forms adauer larva; and (c) contacting said dauer larva with a compound,wherein release from the dauer larval state is an indication that saidcompound is a candidate compound for ameliorating or delaying animpaired glucose intolerance condition.
 6. A method for theidentification of a compound that is a candidate compound forameliorating or delaying an impaired glucose tolerance condition, saidmethod comprising the steps of: (a) providing an age-1, daf-16 mutantnematode; (b) expressing in the cells of said nematode a mammalian AFXpolypeptide, whereby said nematode forms a dauer larva; and (c)contacting said dauer larva with a compound, wherein release from thedauer larval state is an indication that said compound is a candidatecompound for ameliorating or delaying an impaired glucose intolerancecondition.
 7. A method for the identification of a compound that is acandidate compound for ameliorating or delaying an impaired glucosetolerance condition, said method comprising the steps of: (a) providinga daf-2, daf-16 mutant nematode; (b) expressing in the cells of saidnematode a mammalian FKHR polypeptide, whereby said nematode forms adauer larva; and (c) contacting said dauer larva with a compound,wherein release from the dauer larval state is an indication that saidcompound is a candidate compound for ameliorating or delaying animpaired glucose intolerance condition.
 8. A method for theidentification of a compound that is a candidate compound forameliorating or delaying an impaired glucose tolerance condition, saidmethod comprising the steps of: (a) providing an age-1, daf-16 mutantnematode; (b) expressing in the cells of said nematode a mammalian FKHRpolypeptide, whereby said nematode forms a dauer larva; and (c)contacting said dauer larva with a compound, wherein release from thedauer larval state is an indication that said compound is a candidatecompound for ameliorating or delaying an impaired glucose intolerancecondition.
 9. The method of any of claims 5-8, wherein said nematode isC. elegans.
 10. The method of any of claims 5-8, wherein said compoundis a candidate compound for ameliorating or delaying an impaired glucosetolerance condition that involves obesity or atherosclerosis.
 11. Amethod for identifying a compound that modulates the interaction betweenDAF-16 and a second DAF polypeptide, said method comprising the stepsof: (a) providing a DAF-16 polypeptide; (b) providing a second DAFpolypeptide; (c) allowing said DAF-16 polypeptide and said second DAFpolypeptide to interact and form a complex; (c) contacting said complexwith a candidate compound, a modulation in the interaction between saidDAF-16 and said second DAF polypeptide identifying a modulatorycompound.